NOTE:
This is a draft of a general information paper on the history and recent
developments in the field of phage therapy. Any comments and additional
information would be greatly appreciated.
PHAGE THERAPY: BACTERIOPHAGES AS ANTIBIOTICS
Elizabeth
Kutter, Evergreen State College, Olympia, WA 98505 -- Nov. 15, 1997
t4phage@elwha.evergreen.edu;
360 866 6000, X 6099 or 6523
INTRODUCTION
Bacteria resistant to most or all available
antibiotics are causing increasingly serious problems, raising widespread fears
of returning to a pre-antibiotic era of untreatable infections and epidemics.
Despite intensive work by drug companies, no new classes of antibiotics have
been found in recent years. There are hopes that the newfound ability to
sequence entire microbial genomes and to determine the molecular bases of
pathogenicity will open new avenues for treating infectious disease, but other
approaches are also being sought with increasing fervor. One result is a renewed
interest in the possibilities of bacteriophage therapy -- the harnessing of a
specific kind of viruses that attack only bacteria to kill pathogenic
microorganisms (cf. Levin
and Bull, 1996; Lederberg,
1996; Radetsky,
1996; Barrow
and Soothill, 1997).
Phage therapy was first developed early in this
century and showed much promise but also aroused much controversy. It has been
little used in the West since the advent of antibiotics in the 1940s. However,
extensive clinical research and implementation of phage therapy continued to be
carried out in Eastern Europe over the last 50 years. The results of that work
effectively complement the limited recent animal work in the West that is
primarily cited in most recent articles, encouraging optimism that phage can
indeed play an important role in dealing with infections involving increasingly
drug-resistant microbes. We need to draw as much as possible on the
largely-unknown body of knowledge that has accumulated in Poland, France and
many parts of the former Soviet Union (FSU) as we again explore phage therapy,
and to give credit where it is due for the many years of hard, careful work they
have invested in the field. This paper is written primarily to put phage therapy
in historical and ecological context and to explore some of the more interesting
and extensive work in Eastern Europe, little of which has been accessible in
English.
THE NATURE OF BACTERIOPHAGES
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Viruses are like space ships that are able to
carry genetic material between susceptible cells and then reproduce in those
cells, just as the AIDS virus, HIV, specifically infects human T lymphocytes
which carry a particular surface protein called CD4. Each virus consists of a
piece of genetic information, determining all of the properties of the virus,
which is carried around packaged in a protein coat. In the case of
bacteriophages, the targets are specific kinds of bacterial cells; they cannot
infect the cells of more complex organisms because of major differences in key
intracellular machinery as well as in cell-surface proteins. Most phages have
tails, the tips of which have the ability to bind to specific molecules on the
surface of their target bacteria.
The viral DNA is then injected through the tail
into the host cell, where it directs the production of progeny phages -- often
over a hundred in half an hour. Each strain of bacteria has characteristic
protein, carbohydrate and lipopolysaccharide molecules present in large
quantities on its surface. These molecules are involved in forming pores, in
motility, in binding of the bacteria to particular surfaces; each such molecule
can act as a receptor for particular phages. Development of resistance to a
particular phage generally reflects mutational loss of its specific receptor;
this loss often has negative effects on the bacterium and does not protect it
against the many other phage which use different receptors.
Each kind of bacteria has its own phages, which
can be isolated wherever that particular bacterium grows -- from sewage, feces,
soil, even ocean depths and hot springs. The process of isolation is easy. Just
let the sample sit in an appropriate broth, separate off the liquid part, and
pass it through a filter with pores so tiny that bacteria can't get through.
Then add concentrated nutrients, mix it (at several different dilutions) with a
culture of the bacteria in question, and let it incubate under appropriate
growth conditions. Spread a few drops on a block of appropriate nutrient medium
which is made firm with agar taken from seaweed. The next day, one sees a dense
covering or lawn of bacteria with round clear spots, called plaques on plates at
the right dilution. Each plaque contains many million phage particles, all
progeny of one phage which was immobilized there on the agar. That phage
infected a cell, multiplied inside it, and caused it to burst. This released
many phages, which infected nearby cells and repeated the process. One can stick
a toothpick into one of these plaques, transfer it to a fresh culture of the
bacteria in liquid medium, and grow up a homogeneous stock of descendants of
that particular phage, whose properties can then be studied.
HISTORICAL CONTEXT
Discovery
A century ago, Hankin (1896) reported that the waters of the Ganges and Jumna rivers in India had
marked antibacterial action which could pass through a very fine porcelain
filter; this activity was destroyed by boiling. He particularly studied the
effects on Vibrio cholerae and
suggested that the substance responsible was what kept cholera epidemics from
being spread by ingestion of the water of these rivers. However, he did not
further explore the phenomenon. Edward Twort (1915)
and Felix d'Herelle (1917) independently reported
isolating filterable entities capable of destroying bacterial cultures and of
producing small cleared areas on bacterial lawns, seemingly implying that
discrete particles were involved. They are jointly given credit for the
discovery. It was d'Herelle, a Canadian working at the Pasteur Institute in
Paris, who gave them the name "bacteriophages"-- using the suffix
phage not in its strict sense of to eat, but in that of developing at the
expense of (see d'Herelle,
1922, p. 21) - and who made them a major part of his
life's work. D'Herelle, a largely self-trained microbiologist, had just spent 10
years in Guatemala, Mexico and Argentina. There, he dealt with epidemics of
dysentery, yellow fever and a coffee-killing fungus, isolated a bacterium from
dying locusts to use in controlling locust plagues, and explored several
interesting fermentation challenges - all good preparation for his later work
with phages, as discussed in an interesting fashion by Summers (1999). At the
Pasteur Institute, he was carrying out a careful study of vaccine preparation
techniques using a model system - "B. typhimurium" in its natural host, mice; he felt
strongly that meaningful data on immunity and pathogenicity could only be
obtained when natural hosts were used. In his spare time, he was also doing
research with dysentary patients - a frequent problem in wartime France. From
the feces of several of these patients, he isolated a filterable anti-Shiga
"microbe" which multiplied through many serial passages on its host
bacterium, and which could produce tiny clear circles on a plate of this "Shiga
bacillus" (d'Herelle, 1917).
D'Herelle went on to carefully characterize
bacteriophages as viruses which multiply in bacteria and worked out the details
of infection by various phages of different bacterial hosts under a variety of
environmental conditions, always working to combine natural phenomena with
laboratory findings, to better understand immunity and natural healing from
infectious disease (Summers,
1999). The Ninetieth Annual Meeting of the British
Medical Association in Glasgow featured a very interesting discussion between d'Herelle,
Twort and several other eminent scientists of the day on the nature and
properties of bacteriophages (d'Herelle
et al, 1922). The main issue at that time was whether the observed bacteriolytic
principle was an enzyme produced by bacterial activity or a form of tiny virus
with some sort of life of its own, as claimed by d'Herelle; this controversy
continued for many years, splitting the rapidly-growing community of people
working with phages.
D'Herelle summarized the early phage work in a
300-page book "The Bacteriophage" (1922).
He wrote classic descriptions of plaque formation and composition, infective
centers, the lysis process, host specificity of adsorption and multiplication,
the dependence of phage production on the precise state of the host, isolation
of phages from sources of infectious bacteria and the factors controlling
stability of the free phage. He quickly became fascinated with the apparent role
of phages in the natural control of microbial infections. He noted for example
the frequent specificities of the phages isolated from recuperating patients for
their own disease organisms and the rather rapid variations over time in their
phage populations. He thus worked throughout his life to develop the potential
of using properly selected phages as therapeutic agents against the most
devastating health problems of the day. However, he initially focussed on simply
understanding phage biology. Thus, the first known report of successful phage
therapy came not from d'Herelle but from Bruynoghe and Maisin (1921), who used phage to treat staphylococcal skin infections.
After a year at the Pasteur Institute of Saigon,
d'Herelle returned to tight physical conditions, personal conflict and
intellectual controversy at the Pasteur Institute in Paris. He soon accepted an
offer to move to the Netherlands, where he was provided better conditions for
his work with the recovery from infectious disease and the properties of
bacteriophages, published his first book and a number of papers, and received an
honorary MD degree. In 1925, he became a health officer for the League of
Nations, based in Alexandria, Egypt, with special responsibility for controlling
infectious disease on ships passing through the Suez Canal and during some of
the major Muslim pilgrimages. Phage therapy and sanitation measures were the
primary tools in his arsenal to deal with major outbreaks of infectious disease
throughout the Middle East and India. Throughout this period, he continued
publishing on his research and clinical trials and assisting others who were
willing to do so with phages and consultations, often undertaking extended
travel at his own expense. One of the most extensive trials of phage therapy he
helped set up was the Bacteriophage Inquiry of 1927-1936 (Summers,
1993), which led to "what seems to be
convincing results, endorsed by august committees" yet still left many
skeptics of phage therapy; these studies deserve closer scrutiny.
In 1928, d'Herelle was invited to Stanford to
give the prestigious Lane Lectures; his discussion of "The Bacteriophage
and its Clinical Applications" was published as a monograph (d'Herelle and
Smith, 1930). He gave many lectures for medical schools and societies as he
crisscrossed the country. He then went on to Yale to take up a regular faculty
position, arranged with the support of George Smith, who had translated his
first two books into English. He continued to spend summers in Paris working
with the phage company he had established there, run by his son-in-law, in
response to strong demands for phage preparations with careful quality control;
this period is discussed particularly well by Summers (1999). He returned permanently to Europe in 1933, spending much time the
following two years in Tiflis (Tbilisi), Georgia, helping to set up an
international Bacteriophage Institute there, as discussed further below.
From early on, one major practical use of phages
was for bacterial identification through a process called
phage typing -- the use of patterns of sensitivity to a specific battery of
phages to precisely identify microbial strains. This technique takes advantage
of the fine specificity of many phages for their hosts and is still in common
use around the world. The sophisticated ability of phages to destroy their
bacterial hosts can also have a very negative commercial impact; phage
contaminants occasionally spread havoc and financial disaster for the various
fermentation industries that depend on bacteria, such as cheese production and
fermentative synthesis of chemicals (cf. Saunders,
1994)
Phage therapy was tried extensively and many
successes were reported for a variety of diseases, including dysentery, typhoid
and paratyphoid fevers, cholera, and pyogenic (pus-producing) and urinary-tract
infections. Phages were poured directly into lesions, given orally or applied as
aerosols or enemas. They were also given as injections -- intradermal,
intravascular, intramuscular, intraduodenal, intraperitoneal, even into the lung,
carotid artery and pericardium. The early strong interest in phage therapy is
reflected in the fact that some 800 papers were published on the topic between
1917 and 1956; the results have been discussed in some detail by Ackermann and
Dubow (1987). The reported results were
quite variable. Many physicians and entrepreneurs became very excited by the
potential clinical implications and jumped into applications with very little
understanding of phages, microbiology or basic scientific process. Thus many of
the studies were anecdotal and/or poorly controlled, many of the failures were
predictable and some of the reported successes did not make much scientific
sense. Often, uncharacterized phages at unknown concentrations were given to
patients without specific bacteriological diagnosis, and there is no mention of
follow up, controls or placebos.
Much of the understanding gained by d'Herelle
was ignored in this early work, and inappropriate methods of preparation, "preservatives"
and storage procedures were often used. On one occasion, d'Herelle reported
testing 20 preparations from various companies and finding that not one of them
contained active phages (Summers,
1999). On another occasion, a preparation was advertised as containing a
number of different phages, but it turned out that the technician responsible
had decided it was easier to grow them up in one large batch than in separate
batches. Not too surprisingly, checking the product showed that one phage had
outcompeted all the others and this was not, in fact, a polyvalent preparation.
This was the origin of the phage T7, whose RNA polymerase now plays a major role
in biotechnology (William Summers, personal communication). In general, there
was no quality control except in a few research centers. Large clinical studies
were rare and the results of those few were largely inaccessible outside of
Eastern Europe.
In 1931, an extensive review of bacteriophage
therapy was commissioned by the Council on Pharmacy and Chemistry of the
American Medical Association (Eaton
and Bayne-Jones, 1931). Its purpose was "(a)
to present summaries and discussions of (1) the experimentally determined facts
relating to the bacteriophage phenomenon, (2) the laboratory and clinical
evidence for and against the therapeutic usefulness of bacteriophage and (3) the
relation of so-called antivirus to materials containing bacteriophage, and (b)
to serve as a basis for a survey of the status of some of the commercial
preparations." With 150 references, this report made a major effort to
survey at least what they considered the most significant papers and reviews. In
evaluating this report, it is important to realize how little was yet known then
about bacteriophages. In fact, their first conclusion was "Experimental
studies of the lytic agent called "bacteriophage" have not disclosed
its nature. D'Herelle's theory that the material is a living virus parasite of
bacteria has not been proved. On the contrary, the facts appear to indicate that
the material is inanimate, possibly an enzyme." In retrospect, the proof
that phages are viruses looks solid and it is hard to see how they could have
come to this conclusion, which clearly impacted all of their other findings.
These included: "2.) Since it has not been shown conclusively that
bacteriophage is a living organism, it is unwarranted to attribute its effect on
cultures of bacteria or its possible therapeutic action to a vital property of
the substance. 3.) While bacteriophage dissolves sensitive bacteria in culture
and causes numerous modifications of the organisms, its lytic action in the body
is inhibited or greatly impeded by blood and other bodily fluids. 4.) The
material called bacterophage is usually a filtrate of dissolved organisms,
containing, in addition to the lytic principle, antigenic bacterial substances,
products of bacterial growth and constituents of the culture medium. The effects
of all these constituents must be taken into consideration whenever therapeutic
action is tested. 5) A review of the literature on the use of bacteriophage in
the treatment of infections reveals that the evidence for the therapeutic value
of lytic filtrates is for the most part contradictory. Only in the treatment of
local staphylococcic infections and perhaps cystitis has evidence at all
convincing been presented."
This assessment clearly had a strong influence
on the investment of the medical community in exploring phage therapy, at least
in the United States. Points are raised which still need to be considered,
particularly in terms of the many trials described there in animals or humans
which seemed to show little or no success and in terms of such potentially
confounding explanations of the successes as the apparent strong stimulation of
natural immune mechanisms by the bacterial debris in the lysates used. Then in
the 1940's, the new "miracle" antibiotics such as penicillin became
became widely available, and phage therapy was largely abandoned in the western
world.
SPECIFIC PROBLEMS OF EARLY PHAGE THERAPY WORK
Today, many believe that phage therapy was
proven not to work in the early part of this century. However, it appears that
it simply was never given sufficient and appropriate trial, and reassessment is
warranted. It is thus important to consider in some detail potential reasons for
the early problems and the questions as to efficacy. These included:
In making the choice to again explore the
possibilities of phage therapy, we should also consider their many potential
advantages, discussed in more detail below:
PROPERTIES OF PHAGES
One major source of confusion in the early phage
work was the perception that all phages were fundamentally similar, though
subject to adaptive change depending on the recent conditions of growth. One
consequence of this was that often new phages were isolated for each series of
studies, so that there was little continuity or basis for comparison. Phages
specific for over 100 bacterial genera have now been isolated (Ackermann,
1996); they have been found virtually everywhere that they have been sought.
However, only few have yet been well studied or classified (cf. Ackermann
and DuBow, 1987)
A second early source of confusion affecting
therapeutic uses was the question of whether the lytic principle termed "bacteriophage"
simply reflected an inherent property of the specific bacteria or required
regular reinfection by an external agent. During the 1930s and 1940s, it became
increasingly clear that in some senses both were true -- that there were in fact
two quite fundamentally different groups of bacteriophages. Lytic phages always
have to infect from outside, reprogram the host cell and release a burst of
phage through breaking open, or lysing, the cell after a relatively fixed
interval. Lysogenic phages, on the other hand, have another option. They can
actually integrate their DNA into the host DNA, much as HIV can integrate the
DNA copy of its RNA, leading to virtually permanent association as a prophage
with a specific bacterium and all its progeny. The prophage directs the
synthesis of a repressor, which
blocks the reading of the rest of its own genes and also those of any
closely-related lysogenic phages -- a major advantage for the bacterial cell.
Many prophages further aid their host by helping protect against various
unrelated, lytic phages. Occasionally, a prophage escapes from regulation by the
repressor, cuts its DNA back out of the genome by a sort of site-specific
recombination and goes ahead to make progeny phage and lyse open the cell.
Sometimes the cutting-out process makes mistakes and a few bacterial genes get
carried along with the phage DNA to its new host; this process, called
transduction, plays a significant role in bacterial genetic exchange. Such
lysogenic phages are very bad candidates for phage therapy, both due to their
mode of inducing resistance and to the fact that they can potentially lead to
transfer of genes involved in bacterial pathogenicity; this is discussed in more
detail below. However, their specificity often makes them very useful for phage
typing in distinguishing between bacterial strains.
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http://www.evergreen.edu/user/T4/PhageTherapy/manyphi.jpgKey technical developments that helped clarify the general nature and
properties of bacteriophages included: (1) the concentration and purification of
some large phages by means of high-speed centrifugation and the demonstration
that they contained equal amounts of DNA and protein (Schlesinger,
1933 a, b) and (2) visualization of phages by means of the electron microscopic
(EM) (Ruska,1940; Pfankuch
and Kausche, 1940). Soon after, Ruska (1943) reported the first attempts to use the EM for phage systematics; this
has since become a key tool of the field (cf. Ackermann
and DuBow, 1987). Each phage was found to have its own specific shape and size, from the
"lunar lander"-style complexity of T4 and its relatives to the
globular heads with long or short tails of lambda and T7 to the small
filamentous phages that looked much like bacterial pili (See adjacent figure,
from Ackermann,
1996)
LYTIC PHAGES
A much better understanding of the interactions
between lytic phage and bacteria came from detailed one-step growth curve
experiments expanding on the work of d'Herelle (1922)
(Ellis
and Delbrück, 1939, Doermann,1952).
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http://www.evergreen.edu/user/T4/PhageTherapy/infection.jpgThese demonstrated an eclipse period during which the DNA began
replicating and there were no free phage in the cell, a period of accumulation
of intracellular phage, and a lysis process which released the phage to go in
search of new hosts. An example of this phage infection cycle is outlined in the
adjacent figure (courtesy of Chris Mathews, OSU).
In the early 1940's, developments occurred which
were to have a major impact on the orientation of phage research in the United
States and much of western Europe, strongly shifting the emphasis from practical
applications to basic science. Physicist-turned-phage-biologist Max Delbrück
began working with key phage biologists Alfred Hershey and Salvador Luria and
formed the "Phage Group", which eventually expanded dramatically with
aid of the summer Phage Courses at Cold Spring Harbor, Long Island. These ran
for many years starting in 1945 and regular phage meetings still continue there,
although they have now shifted their focus to "phage and microbial genetics."
The influence of the Phage Group on the origins
of molecular biology has been well documented (cf. Cairns
et al, 1966; Fischer
and Lipson, 1988; Summers,
1993b). Virulent phages had just the right balance of complexity and
simplicity to tease out the key concepts of cell regulation at the molecular
level. However, a major element of the rapid success of phage as model systems
was that Delbrück convinced most phage biologists in the United States to focus
on one bacterial host (E. coli B) and
7 of its lytic phages, building a very strong, tightly focussed community all
working on the same set of problems, able to build effectively on each other's
work and communicate easily. The 7 phages were arbitrarily chosen and named
T(type)1-T7. As it turned out, T2, T4 and T6 were quite similar to each other,
defining the "T-even" family of phages, discussed in more detail below.
These phages were key in demonstrating that DNA is the genetic material, that
viruses can encode enzymes, that gene expression is mediated through special
copies in the form of "messenger RNA", that the genetic code is
triplet in nature, and many other fundamental concepts. The negative side of
this strong focus on a few phages growing under rich laboratory conditions,
however, was that there was very little study or awareness of the ranges, roles
and properties of bacteriophages in the natural environment, or of phages that
infect other kinds of bacteria.
RATIONAL PHAGE THERAPY
The rapid, powerful developments in the
understanding of phage biology had the potential to facilitate more rational
thinking about the therapeutic process and the selection of therapeutic phages.
However, there was generally little interaction between those who were so
effectively using phage as tools to understand molecular biology and those still
working on phage ecology and therapeutic applications. Many in the latter group
were spurred on by concern about the increasing incidence of nosocomial (hospital-acquired)
infections and of bacteria resistant against most or all known antibiotics. This
is particularly true in Poland, France and the former Soviet Union where use of
therapeutic phages never fully died out and where there has been extensive
ongoing research and clinical experience. In France, Dr. Jean-François Vieu led
the therapeutic phage efforts until his retirement some 10 years ago. He worked
in the "Service des Entérobactéries" of the Pasteur Institute in
Paris and, for example, prepared Pseudomonas phages on a case-by-case basis for
patients. The experience there is discussed in Vieu (1975) and Vieu et al. (1979). Phage therapy was used extensively in many parts of Eastern Europe as
a natural part of clinical practice, and there are now companies in Moscow and
several other Russian cities making phage preparations for this purpose. However
most of the research and much of the phage preparation came under the direction
of key centers in Tbilisi, Georgia and in Wroclaw, Poland. I will thus focus on
the work of these two groups.
Polish
Academy of Sciences, Wroclaw
The most detailed publications documenting phage
therapy have come from Stefan Slopek's group at the Institute of Immunology and
Experimental Medicine of the Polish Academy of Sciences in Wroclaw. This group
published a series of detailed papers in the Archivum Immunologiae et Therapie
Experimentalis (cf. Slopek
et al, 1983, 1985, 1987), describing the results of phage treatments carried out from 1981 to
1986 with 550 patients. This set of studies involved ten Polish medical centers,
including the Wroclaw Medical Academy Institute of Surgery Cardiosurgery Clinic.
Children's Surgery Clinic and Orthopedic Clinic; the Institute of Internal
Diseases Nephrology Clinic and Clinic of Pulmonary Diseases. The patients ranged
in age from 1 week to 86 years; in 518 of the cases, phage use followed
unsuccessful treatment with all available antibiotics. The major categories of
infections treated were long-persisting suppurative fistulas, septicemia,
abscesses, respiratory tract suppurative infections and bronchopneumonia,
purulent peritonitis and furunculosis. In a final summary paper (Slopek
et al, 1987), the authors carefully analyzed the results with regard to such factors
as nature and severity of the infection and monoinfection vs. infection with
multiple bacteria. Rates of success ranged from 75 to 100 % (92% overall), as
measured by marked general improvement of health, tendency to heal of local
wounds and disappearance of titratable bacteria; 84% demonstrated full
elimination of the suppurative process and healing of local wounds. Infants and
children did particularly well; not surprisingly, the poorest results came with
the elderly and those in the final stages of extended serious illness, with
weakened immune systems and generally poor resistance.
The bacteriophages used all came from the
extensive collection of the Bacteriophage Laboratory of the Institute of
Immunology and Experimental Therapy; in the later studies, some of the specific
phages used were named. All were virulent, capable of completely lysing the
bacteria being treated. In the first study alone, 259 different phages were
tested (116 for Staphylococcus, 42 for Klebsiella, 11 for Proteus, 39 for
Escherichia, 30 for Shigella, 20 for Pseudomonas, and one for Salmonella); 40%
of them were selected to use directly for therapy. All of the treatment was in a
research mode, with the phage prepared at the Institute by standard methods and
tested for sterility. Treatment generally involved 10 ml of sterile phage lysate
orally half an hour before each meal, with gastric juices neutralized by (basic)
Vichy water, baking soda or gelatin. In addition, phage-soaked compresses were
generally applied three times a day where dictated by localized infection.
Treatment ran for 1.5-14 weeks, with an average of 5.3; for intestinal problems,
short treatment sufficed, while it was very long for such problems as pneumonia
with pleural fistula and pyogenic arthritis. Bacterial levels and phage
sensitivity were continually monitored, and the phage(s) being used were changed
if the bacteria lost their sensitivity; therapy was generally continued for two
weeks beyond the last positive test for the bacteria.
Few side effects were observed; those that were
seen seemed directly associated with the therapeutic process. Pain in the liver
area was often reported around day 3-5, lasting several hours; the authors
suggested that this might be related to extensive liberation of endotoxins as
the phage were destroying the bacteria most effectively. In severe cases with
sepsis, patients often ran a fever for 24 hours about days 7-8 (Slopek
et al, 1981a). Various other methods of administration were successfully used,
including aerosols and infusion rectally or in surgical wounds. Intravenous
administration was not recommended for fear of possible toxic shock from
bacterial debris in the lysates (Slopek
et al, 1981a). However, it was clear that the phages readily got into the body from
the digestive tract and multiplied internally wherever appropriate bacteria were
present, as measured by their presence in blood and urine as well as by
therapeutic effects (Weber-Dabrowska
et al, 1987).
Detailed notes were kept throughout on each
patient. The final evaluating therapist also filled out a special inquiry form
that was sent to the Polish Academy of Science research team along with the
notes. The Computer Center at Wroclaw Technical University carried out the
extensive analyses of the data. The authors used the categories established in
the WHO (1977) International Classification of Diseases in assessing results.
They also looked at the effects of age, severity of initial condition, type(s)
of bacteria involved, length of treatment and other concomitant treatments. The
papers include many specific details on individual patients which help give
insight into the ways phage therapy was used, as well as an in-depth analysis of
difficult cases.
Bacteriophage
Institute, Tbilisi
The most extensive and least widely known work
on phage therapy was carried out under the auspices of the Bacteriophage
Institute at Tbilisi, d'Herelle's institute in the former Soviet republic of
Georgia. The work there will thus be discussed in some detail.
Georgia is an ancient and beautiful country of 5
million people, lying tucked in a valley between the High Caucasus mountains at
the south of Russia, the Samtsxe-Dgavaxete range bordering on Turkey and
Armenia, and the Black Sea. Through all the centuries of political upheaval at
this crossroads of the ancient world, it has managed to keep its own culture and
unique language, which is related only (and remotely) to Basque. It has been
Christian since the third century, but prides itself strongly on its openness to
all religions and cultures, its synagogue, mosque, and various Christian
churches all clustered in the heart of old Tbilisi. Strong emphasis is placed on
culture, intellectual pursuits and hospitality; the literacy rate is 100%,
according to the 1996 UN Human Development Report on Georgia, and it has a long
tradition of excellence in fields from music to mathematics to wine-making and
cooking.
According to various Georgian physicians with
whom I have spoken, phage therapy is part of the general standard of care there,
used especially extensively in pediatric, burn and surgical hospital settings.
Phage preparation was carried out on an industrial scale, employing 1,200 people
just before the break-up of the Soviet Union. Tons of tablets, liquid
preparations and spray containers of carefully-selected mixtures of phages for
therapy and prophylaxis were shipped throughout the former Soviet Union (FSU)
each day. They generally were available both over the counter and through
physicians. The largest use was in hospitals, to treat both primary and
nosocomial infections, alone or in conjunction with chemical antibiotics. They
played a particularly important role when antibiotic-resistant organisms were
found. The military is still one of the strongest supporters of phage therapy
research and development, because phages have proven so useful for wound and
burn infections as well as for preventing debilitating gastrointestinal
epidemics among the troops.
The historical background of the institute is
interesting, and reflects a relatively unknown period in d'Herelle's caeer. The
following material comes from a number of people at the Institute, from a recent
article by Shrayer (1996) on d'Herelle in Russia, from Summers (1998)
and from d'Herelle's own work.
In 1917, George Eliava, of the Georgian
Institute of Microbiology, noticed that the water of the Koura (Mtkvary) river
in Tbilisi (Tiflis) had a bactericidal action - an observation that could be
explained by d'Herelle's bacteriophage discovery. Eliava spent several extended
periods in Paris at the Pasteur Institute and was a very early and staunch
collaborator of d'Herelle's; several papers of his are cited in d'Herelle's
first book on phages (1922). The two developed the
dream of founding an Institute of Bacteriophage Research in Tbilisi, to be a
world center of phage therapy for infectious disease, including scientific and
industrial facilities and supplied with its own experimental clinics. The dream
quickly became a reality due to the support of Sergo Ordjonikidze, the People's
Commissar of Heavy Industry, despite KGB opposition to this "foreign
project" and personal conflicts between Eliava and Beria, then the Georgian
KGB head who was soon to direct the Soviet KGB as Stalin's much-feared henchman.
A large campus on the river Mtkvary was allotted for the project in 1926. For
many years, d'Herelle sent supplies, equipment and library materials, most of
which he paid for himself. In 1934 -1935 he and his wife spent two 6-month
periods working in Tbilisi, during which time he visited Kamenski, the People's
Commisar of Health Care, in Moscow and turned down an invitation to move there.
He also wrote a book on "The Bacteriophage and the Phenomenon of Recovery",
which was translated into Russian by Eliava and dedicated to Stalin. D'Herelle
intended to eventually move to Georgia; in fact, the cottage built for his use
still stands on the Institute grounds. However, in 1937 Eliava was arrested as a
"People's Enemy" by Beria. Eliava was summarily executed without a
trial, sharing the tragic fate of many Georgian and Russian progressive
intellectuals of the time, and d'Herelle, disillusioned, never returned to
Georgia or the USSR. However, their Institute survived, and is still functioning
at its original site on the Mtkvary (which it now shares with the more modern
Institutes of Molecular Biology and Biophysics and of Animal Physiology).
In 1938, the Bacteriophage Institute was merged
with the Institute of Microbiology and Epidemiology, under direction of the
People's Commissary of Health of Georgia. In 1951, it was formally transferred
to the All-Union Ministry of Health set of Institutes of Vaccines and Sera,
taking on the leadership role in providing bacteriophages for therapy and
bacterial typing throughout the former Soviet Union. Under orders from the
Ministry of Health, hundreds of thousands of samples of pathogenic bacteria were
sent to the Institute from throughout the Soviet Union, to isolate more
effective phage strains and better characterize their usefulness. In 1988, the
Scientific Industrial Union "Bacteriophage" was formed, centered in
Tbilisi with Russian production facilities in Ufa, Khabarowsk and Nijnyi
Novgorod. The industrial part was always run on a self-supporting basis. The
institute's government-supported scientific branch included the electron
microscope facility, permanent strain collection, laboratories studying phages
of the enterobacteria, staphylococci and pseudomonads and formulating new phage
cocktails, and groups involved in immunology, vaccine production, work with Lactobacillus
and other therapeutic approaches. It also carried out the very extensive studies
needed for approval by the Ministry of Health in Moscow of each new phage strain,
therapeutic cocktail and means of delivery.
This careful study of the host range, lytic
spectrum and cross-resistance properties of the phages being used were a major
factor in the reported successes of the phage therapy work carried out through
the Institute. All of the phages used for therapy are lytic, avoiding the
problems engendered by lysogeny. The problems of bacterial resistance were
largely solved by the use of well-chosen mixtures of phages with different
receptor specificities against each type of bacterium as well as of phages
against the various bacteria likely to be causing the problem in multiple
infections. The situation was further improved whenever the clinicians typed the
pathogenic bacteria and monitored their phage sensitivity; where necessary, new
cocktails were then prepared to which the given bacteria were sensitive. Not
infrequently, using phage in conjunction with other antibiotics was shown to
give better results than either the phage or the antibiotic alone.
The depth and extent of the work involved is
very impressive. For example, in 1983-85 alone, the Institute's Laboratory of
Morphology and Biology of Bacteriophages carried out studies of growth,
biochemical features and phage sensitivity of 2038 strains of Staphylococcus,
1128 of Streptococcus, 328 of Proteus, 373 of Ps. aeruginosa and 622 of
Clostridium, received from clinics and hospitals in towns across the former
Soviet Union. New broader-acting phage strains were isolated using these and
other Institute cultures and included in a reformulation of their
extensively-used Piophage preparation; it now inhibited 71% of their
Staphylococcus strains instead of 58%, 76% of Pseudomonas instead of 55%, 51% of
E. coli instead of 11%, 30% of Proteus instead of 3%, 60% of Streptococcus
instead of 38%, and 80% of Enterococcus instead of 3% (Zemphira
Alavidze, personal communication.) In the years since, there have been continued
improvements in the formulation based on further studies, and phages against
Klebsiella and Acinetobacter have been isolated and developed into therapeutic
preparations. One of the latest developments is their IntestiPhage preparation,
which includes 23 different phages active against a range of enteric bacteria.
A good deal of work has gone into developing and
providing the documentation to get approval from the Ministry of Health for
specialized new delivery systems, such as a spray for use in respiratory-tract
infections, in treating an incision area before surgery, and in sanitation of
hospital problem areas such as operating rooms. An enteric-coated pill was also
developed, using phage strains that could survive the drying process, and
accounted for the bulk of the shipments to other parts of the former Soviet
Union. Phage are particularly effective when poured or sprayed directly into
wounds or other major sites of infection, since they can first multiply in
bacteria near the surface and then reach deeper and deeper sites in subsequent
cycles of infection. For example, the attached figure shows the use of phage to
treat a severe foot infection. This diabetic patient came in to have his foot
amputated. Instead, doctors split the foot open to facilitate access and poured
in phage. When the dressing was removed several days later, all signs
of purulence (or pussy infection) were gone.
Much of the focus in the last 12 years has been
on combating nosocomial infections, where multi-drug-resistant organisms have
become a particularly lethal problem and where it is also easier to carry out
proper long-term research. Clinical studies of the effectiveness of the phage
treatment and appropriate protocols were carried out in collaboration with a
number of hospitals, but little has been published in accessible form. Zemphira
Alavidze and her colleagues who are currently doing most of the actual
therapeutic development and clinical application have manuscripts in preparation
which describe their work in institutions such as the Leningrad (St. Petersburg)
Intensive Burn Therapy Center, the Academy of Military Medicine in Leningrad,
the Kazan Trauma Center, the Kemerovo Maternity Hospital. Some of the most
intensive studies were carried out in Tbilisi, at the Pediatric Hospital, the
Burn Center, the Center for Sepsis and the Institute for Surgery. Special
mixtures were developed for dealing with strains giving problems of nosocomial
infections in various hospitals, and they were very effectively used in
sanitizing operating rooms and equipment, water taps and other sources of spread
of the infections (most of them involving predominantly Staphylococcus). (Table
1).
The Industrial Branch on the grounds of the
Bacteriophage Institute had huge vats for growing the selected phage, using
appropriate nonpathogenic bacteria and broth they prepared themselves from
high-quality beef. The resulting phage lysates were sterile filtered using
ceramic filters which could themselves be sterilized in very hot ovens. The
various different phages for each particular formulation were then combined and
automatically packaged and sealed into 10-ml ampoules or otherwise prepared and
packaged for administration. Approximate titers were determined by checking the
dilution that would produce lysis after coinnoculation with specific numbers of
bacteria of standard test strains, and each batch was also tested for any
surviving bacterial contaminants. In those rare cases where injection was
planned, the phages were concentrated and resuspended in physiological saline
solution; testing in guinea pigs was added to the rest of the analytical regime,
to make sure there were no residual bacterial surface fragments (endotoxins)
that might cause problems if injected. (As mentioned above, phages have
generally been reported to appear in the bloodstream and other body fluids
rather shortly after being ingested or poured into a wound and to still be
effective against systemic infections, so injection is usually not necessary.)
Injectable forms accounted for only about 5% of
the phage production at its height at the Bacteriophage Institute. None are made
there now due to such factors as the expense and complexities of keeping animals
for the necessary toxicity controls in the difficult situation in Georgia since
the dissolution of the Soviet Union. The extensive fighting in Abkhasia left
350,000 refugees in a country of 5 million people and cut off the major rail,
road and power routes to Russia, leaving only one significant highway across the
High Caucausus mountains. Power is still often available for only a few hours a
day and heating is a serious problem in winter. The country has very little
money available for science, but some research continues, despite virtually no
funding for salaries or supplies. The conflict did provide an interesting
opportunity for widespread phage use. Each soldier in the Georgian army carried
a spray-on phage cocktail which they used to disinfect their wounds (Alavadze,
Meipariani, Gvasalia, manuscript in preparation). The industrial plant was
privatized a few years ago and put to other uses, so the phages currently used
for therapy must be grown in large carboys, the appropriate mixtures made, and
then transferred to vials and sealed by hand. However, the checks for sterility
and efficacy on the designated bacteria are still just as careful.
Unfortunately, until the electron microscope is repaired and electricity made
more predictable, the phage preparations can no longer be checked to be sure
that the phage present are of the appropriate mixtures of morphotypes, or
physical shape and size. The Institute scientists still continue to do the best
they can under the circumstance, and many in Tbilisi feel they clearly owe their
lives to the group's efforts. Extensive therapeutic work still goes on in local
surgical, burn, pediatric and infectious disease hospitals, and in local clinics
for ambulatory patients, including one on the grounds of the Institute.
RECENT WORK IN THE WEST RELATED TO PHAGE THERAPY
Levin and Bull (1996)
and Barrow and Soothill (1997) have provided good reviews of much of the work applying phage therapy
in animals which has been carried out in Britain and the United States since
interest in the possibilities of phage therapy began to resurface there in the
early 80's. The results in general are in very good agreement with the clinical
work described above in terms of efficacy, safety and importance of appropriate
attention to the biology of the host-phage interactions, reinforcing trust in
the reported extensive eastern European results.
In Britain, H. W. Smith and M. B. Huggins (1982, 1983) carried out a series of studies on use of phages in systemic E. coli
infections in mice and then in diarrheal disease in young calves. For example,
they found that injecting 106 colony-forming units of a particular
pathogenic strain intramuscularly killed 10/10 of the mice, but none died if
they simultaneously injected 104 plaque-forming units of a phage
selected against the K1 capsule antigen of that bacterial strain.This phage
treatment was more effective than using such antibiotics as tetracycline,
streptomycin, ampicillin or trimethoprim/sulfafurazole. Furthermore, the
resistant bacteria that emerged had lost their capsule and were far less
virulent. In calves, they found very high levels of protection even though they
did not succeed in isolating phages specific for the K88 or K99 adhesive
fimbriae, which play key roles in attachment within the small intestine. Still,
the phage were able to reduce the number of bacteria bound there by many orders
of magnitude and to virtually stop the fluid loss. The results were particularly
effective if the phage were present before or at the time of bacterial
presentation, and if multiple phages with different attachment specificities
were used. Furthermore, the phage could be transferred from animal to animal,
supporting the possibility of prophylactic use in a herd. If phage were given
only after the development of diarrhea, the severity of the infection was still
substantially reduced and none of the animals died (Smith
et al, 1987). Levin and Bull (1996) carried out a detailed analysis of the population dynamics and tissue
phage distribution of the 1982 Smith and Huggins study which can be helpful in
assessing the parameters involved in successful phage therapy and its apparent
superiority to antibiotics. They have gone on to do very interesting animal
studies of their own (Levin and Bull, manuscript in preparation) and conclude
that phage therapy is at least well worth further study.
Soothill (1994) carried out a series of very
nice studies preparatory to using phages for infections of burn patients. Using
guinea pigs, he showed that skin-graft rejection could be prevented by prior
treatment with phage against Pseudomonas aeruginosa. He also saw excellent
protection of mice against systemic infections with both Pseudomonas and
Acinetobacter when appropriate phages were used (Soothill, 1992). In the latter
case, as few as 100 phages protected against infection with 100 million bacteria
-- 5 times the LD50!
Merrill and coworkers (1996) have carried out a series of experiments designed to better understand
the interactions of phages with the human immune system. Merrill acted as a
primary consultant to Dr. Richard Carlton in starting a company called
"Exponential Biotherapies, Inc." to explore the possibilities of phage
therapy. Their collaborative published work has been with a lytic derivative of
the lysogenic phage lambda. While this particular strain would be a poor choice
for therapeutic use, as discussed above and below, they have gathered very
interesting and important data about factors affecting interactions between
phages and the immune system.
BACTERIAL PATHOGENICITY
Most bacteria are not pathogenic; in fact, they
play crucial roles in the ecological balance in various parts of our bodies,
including the digestive system and all body surfaces. They actually help protect
us from pathogens; this is one reason why the use of broad-spectrum antibiotics
leaves us so vulnerable, and why more narrowly-targeted bactericidal agents
would be highly advantageous. Furthermore, most of the serious pathogens are
close relatives of non-pathogenic strains -- so what are the differences that
make particular strains so lethal? Studies clarifying the mechanisms of
pathogenesis at the molecular level have progressed remarkably in recent years
(cf. Falkow
1996). These have now been crowned by the determination of the complete DNA
base sequence of (nonpathogenic) E. coli K12 and several other bacterial species
and extensive cloning and sequencing of pathogenicity determinants. Generally, a
number of genes are involved, and these are clustered in so-called
"pathogenicity islands", or "Pais", which may be
50,000-200,000 base pairs long. They generally have some unique properties
indicating that the bacterium itself probably acquired them as a sort of
"infectious disease" at some time in the past, and then kept them
because they helped the bacterium infect new ecological niches where there was
less competition. Many of these Pais are carried on small extrachromosomal
circles of DNA called plasmids, which also can be carriers of drug-resistance
genes. Others reside in the chromosome; there, they often are found imbedded in
defective lysogenic prophages which have lost some key genes in the process and
cannot be induced to form phage particles. However, they sometimes can recombine
with related infecting phages. Therefore, it makes sense to avoid using
lysogenic phages or their lytic derivatives for phage therapy to avoid any
chance of picking up and moving such pathogenicity islands.
For bacteria in the human gut, pathogenicity
involves 2 main factors: (1) the production of toxin molecules, such as shiga
toxin (from Shigella and some pathogenic E. coli) or cholera toxin. These toxins
modify proteins in the target host cells and thereby cause the problems. (2) the
acquisition of new cell-surface adhesins which allow the bacterium to bind to
specific receptor sites in the small intestine, rather than just moving on
through to the colon. They also all contain the components of so-called type-III
secretion machinery, related to those involved in assembly of flagella (for
motility) and of filamentous phages and instrumental in many plant pathogens.
For all of the pathogenic enteric bacteria, the infection process triggers
changes in the neighboring intestinal cells. These include degeneration of the
microvilli, formation of individual "pedestals" cupping each bacterium
and, in the case of Salmonella and Shigella, induction of cell-signaling
molecules that trigger engulfment of the bacterium and its subsequent growth
inside the cell.
Recently, E. coli O157 has been the subject of much concern, with contamination
of such products as hamburgers and unpasteurized fruit juices leading to serious
epidemics (cf. Grimm
et al., 1995). Deaths have occurred,
particularly in young children and the elderly, usually from hemorrhagic colitis
(bloody diarrhea) or hemolytic-uremic syndrome, where the kidneys are affected.
Antibiotic therapy has shown no benefit (cf. Greenwald
and Brandt, 1997). We find that the version of O157 from the Seattle fast-food-chain
epidemic, at least, is susceptible to several of our T4-related phages (Mark
Mueller, Kutter et al., unpublished). It is interesting to consider their
potential use in feedlots and meat-packing plants and in prophylaxis and therapy
during outbreaks.
THE T-EVEN FAMILY OF PHAGES
A substantial fraction of the phages in
therapeutic mixes are relatives of bacteriophage T4, which has played such a key
role in the development of molecular biology (cf. Karam, 1994). As discussed
above, the name "T-even family of phages" is a historical accident
reflecting the fact that T2, T4 and T6 out of the original collection of Delbrück's
"Phage Group" all turned out to be related. Large sets of T4-like
phages have been isolated for study from all over the world -- for example, from
Long Island sewage treatment plants, animals in the Denver zoo, and dysentery
patients in Eastern Europe (the latter often using Shigella as host). Members of
the family are found infecting most enteric bacteria and their relatives (Ackermann
and Krisch, Archives of Virology, in
press). Most of the T-even phages studied to date use 5-hydroxymethylcytosine
instead of cytosine in their DNA, which protects them against most of the
restriction enzymes bacteria make to protect themselves against invading DNA and
gives them a much more effective host range. The entire DNA base sequence of
phage T4 is known (Kutter, Stidham et al., 1994)
and we know a great deal about its infection process in standard laboratory
conditions and about the methods it uses to so effectively target bacteria. We
can potentially use some of that knowledge in developing more targeted
approaches to phage therapy, particularly as more is learned about the
similarities and differences in its extended family (cf. Monod
et al., 1997; Kutter
et al., 1996.) We know that different members of the T-even family use different
outer membrane proteins and oligosaccharides as their receptors, and understand
the tail-fiber structures involved well enough to potentially predict which
phages will work on given bacteria and engineer phages with new specificities
(cf. Henning and Hashemolhosseini, 1994; Krisch, personal communication.)
There have still been far too few studies of T4
ecology and its behavior under conditions more closely approaching the natural
environment and the circumstances it will encounter in phage therapy, where the
environment is often anaerobic and/or the bacteria experience frequent periods
of starvation. The limited available information in that regard was summarized
by Kutter, Kellenberger et al (1994).
A variety of studies are shedding light on the ability of these highly virulent
phages to coexist in balance with their hosts in nature. For example, they can
reproduce in the absence of oxygen as long as their bacterial host had been
growing anaerobically for several generations. We have found that they can also
survive for a period of time in a sort of state of hibernation inside of starved
cells and then allow their host to readapt enough when nutrients are again
supplied to go on and produce a few phage. This is particularly interesting and
important since bacteria undergo many drastic changes to survive periods of
starvation which increase their resistance to a variety of environmental insults
(cf. Kolter,
1992).
The T-even bacteriophages share a unique ability
that contributes significantly to their widespread occurrence in nature and to
their competitive advantage. They are able to control the timing of lysis in
response to the relative availability of bacterial hosts in their environment.
When E. coli cells are singly infected with T4, they lyse after 25-30 minutes at
body temperature in rich media, releasing about 100-200 phage per cell. However,
when additional T-even phages attack the cell more than 4 minutes after the
initial infection, the cell does not lyse at the normal time. Instead, it
continues to make phage for as long as 6 hours, with the exact time of eventual
lysis affected by the multiplicity of superinfecting phage (cf. Doermann,
1948; Abedon,
1994). This delay is termed "lysis
inhibition".
Thus, for many reasons the T4-related family of phages make excellent candidates
for therapeutic use in enteric and other gram-negative bacteria, and studies of
their ecology and distribution are being carried out with these goals in mind
both in Tbilisi and at The Evergreen State College. Developing this same sort of
understanding of other phage families potentially useful in phage therapy is
equally important, taking advantage of the many powerful tools now available.
Work useful to this end is progressing in a number of labs around the world but
is still in its infancy, particularly as one moves beyond the enteric bacteria.
CONCLUSIONS
It is clearly time to look more carefully at the
potential of phage therapy, both through strongly supporting new research and
examining carefully what is already available. As Barrow and Soothill conclude,
"Phage therapy can be very effective in certain conditions and has some
unique advantages over antibiotics. With the increasing incidence of antibiotic
resistant bacteria and a deficit in the development of new classes of
antibiotics to counteract them, there is a need to investigate the use of phage
in a range of infections." The stipulations of Ackermann (1987) are important here: "Blind treatment is clearly of no value;
phages have to be tested just as antibiotics, and the indications have to be
right, but this holds everywhere in medicine. However, phage therapy requires
the creation of phage banks and a close collaboration between the clinician and
the laboratory. Phages have at least one advantage....While the concentration of
antibiotics decreases from the moment of application, phage numbers should
increase. Another advantage is that phages are able to spread and thus prevent
disease. Nonetheless, much research remains to be done ... on the stability of
therapeutic preparations; clearance of phages from blood and tissues; their
multiplication in the human body; inactivation by antibodies, serum or pus; and
the release of bacterial endotoxins by lysis... In addition, therapeutic phages
should be characterized at least by electron microscopy." While it seems
premature to generally introduce injectible phage preparations in the West
without further extensive research, their carefully-implemented use for a
variety of agricultural purposes and in external applications could potentially
help reduce the emergence of antibiotic-resistant strains. Furthermore,
compassionate use of appropriate phages seems warranted in cases where bacteria
resistant against all available antibiotics are causing life-threatening
illness. They are especially useful in dealing with recalcitrant nosocomial
infections, where large numbers of particularly vulnerable people are being
exposed to the same strains of bacteria in a closed hospital setting. In this
case, the environment as well as the patients can be effectively treated.
In 1925, Sinclair Lewis's classic novel
Arrowsmith, for which he won the Nobel prize in literature, played a significant role
in raising popular interest in the possibilities of phage therapy and the
potential scientific and ethical dilemmas involved (Summers,
1991). Today, the growing scientific, public and commercial interest in phage
therapy is being reflected and fanned in a number of ways. For example, the BBC
recently produced a Horizon documentary on phage therapy, The
Virus
that Cures, building on the ideas in Radetsky's Discover article on Return of the
Good Virus. Several companies are beginning to explore work with phage therapy.
In addition, a nonprofit "PhageBiotics" foundation has been formed to help support communication,
education and research in the field. Hopefully all of this attention will lead
to increased support of badly-needed research in the field and to rapid progress
in developing appropriate applications, providing at least one alternative to
the growing problem of multi-drug-resistant bacteria.
Acknowledgments:
Special thanks to Drs. Rezo Adamia, Zemphira Alavidze, Teimuraz and Nino
Chanishvili, Taras Gabisonia, Liana Gachechiladze, Mzia Kutateladze, Amiran
Meipariani and their colleagues at the Bacteriophage Institute, Tbilisi, for
their hospitality and efforts to help me understand the extensive therapeutic
work carried out there. Others who have been particularly helpful with
information and communication include Dr. Marina Shubladze, pediatrician in
Tbilisi for 10 years, now residing in Seattle; Nino Mzavia, Nino Trapaidze,
Timur and Natasha Zurabishvili, who have worked in my laboratory on basic T4
biology; Hans-Wolfgang Ackermann (Laval University), Eduard Kellenberger
(Basel), William Summers (Yale), Steve Abedon (Ohio State) and Bruce Levin
(Emory); Mansour Samadpour, University of Washington; Kathy d'Acci (clinical lab
director, St. Peter's Hospital, Olympia); physicians Jess Spielholz, MD, and
Robin Moore, ND; and, especially, the many colleagues and students involved in
our laboratory at Evergreen, particularly Barbara Anderson, Pia Lippincott, Mark
Mueller, Stacy Smith, Elizabeth and Chelsea Thomas, Burt Guttman and Jim
Neitzel.
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The
Virus That Cures, a BBC Horizon Documentary produced by Judith Bunting.
