Journeys in Medicine and Research on Three Continents Over 50 Years

The following sections are included:

• Dedication
• Epigraph
• Preface
• Contents
• Acknowledgements

At this distance in miles, and in time (more than seventy years) what images arise as I think of South Africa? There are vistas of open veldt, grassy plains with occasional flat-topped thorn trees and blue mountains at the edge of the horizon. There are images of wild flowers, aloes with fleshy leaves and orange, red and yellow blossoms on tall erect stems.

My ambitions to become a doctor were further fueled during my years in high school by the many books I read, including Paul de Kruif’s Microbe Hunters, and the biography of Harvey Cushing, neurosurgeon. I read novels by A.J. Cronin and Axel Munthe; both of these authors were physicians and their novels portrayed the struggles and joys of being a physician.

After graduating from medical school in 1963 I took a position as an intern in surgery at Baragwanath Hospital, which served the people of Soweto. South Africa was racially segregated at the time and Soweto was a sprawling suburb for “non-whites” located about forty miles from Johannesburg. Soweto accommodated many people who had come from rural areas to find work in the city. Housing was for the most part inadequate. The streets were poorly lit and the roads were dusty tracks. Entertainment and sports facilities were almost non-existent then. Beer parlors “shebeens”, where a potent homebrewed beer was sold, were plentiful. Particularly at the end of each month when workers were paid, gang members accosted them on their way home and frequently used violent methods to relieve workers of their meager earnings.

In a comprehensive paper on “Urbanization and Development in South Africa”, Ivan Turok (2012) noted: “For over a century urbanization has been a source of controversy, posing dilemmas for successive governments resulting in wide ranging intervention to control it in various ways” .

There are two particular cases that are still embedded in my memories of time spent as an intern in Obstetrics in South Africa in 1964. Both cases came in as patients to a small mission hospital located at some distance from Pretoria.

In January 1966, I started work as a temporary house officer in the Department of Pediatrics in Hospitals affiliated with Glasgow University, Scotland. My first assignment was at the Queen Mother’s Maternity Hospital. Our main duties as house officers included care of infants in a unit specially dedicated to the care of premature infants and newborns at risk due to problems that were detected at birth or early after birth.

My own encounters with neural tube defects occurred in two locations very distant from each other. As a young physician employed as a house officer at the Queen Mother’s hospital in Glasgow, Scotland, ward round duties for a short time included a visit to a single infant in a separate room located distant from the regular nurseries. This infant was born with anencephaly, a form of neural tube defect where the top of the skull and brain had not developed properly. Anencephaly frequently leads to stillbirth; anencephalic infants born alive die within a few days or weeks. I was deeply moved by the attention the nurses gave to this infant in the isolated room. One nurse told me that they had been instructed not to feed the anencephalic infant. However, the infant made soft sounds and when offered bottle feed it seems the infant sucked for brief periods. The parents of this infant were deeply distressed as they waited for the inevitable death of their child.

My most memorable experience of thyroid hormone deficiency occurred when I was a house officer at the Yorkhill Children’s Hospital in Glasgow. Three siblings were admitted to the hospital at the same time. The eldest, a girl of 12 years of age, manifested symptoms of severe thyroid hormone deficiency: growth retardation, intellectual impairments, and thickening of the tissues of the face and neck. These features had in previous decades been referred to as manifestations of cretinism. The second sibling, a girl of nine years of age, had evidence of growth retardation and some evidence of developmental delay. The youngest sibling, a boy of five years of age, had growth delay. On laboratory testing, all three children were found to have thyroid hormone deficiency. They were children of a couple who travelled around in a caravan and who had no fixed address. The children did not have regular medical care and they did not attend school. The three siblings were devoted to each other. Although they each had separate beds in the hospital ward, we would regularly find them huddled together in one bed if we entered the ward late at night or early in the morning. The children were thought to have a defect in a biochemical process that leads to iodination of thyroid hormone in the thyroid gland. Their deficiency of active thyroid hormone could be readily compensated for with treatment using thyroid hormone pills. It was also particularly important to gain the parents’ confidence in order to persuade them to work with us, and subsequently with district nurses and facilities where they located to, in order to ensure ongoing care of the children and continued access to the required medication.

I think back to my school years and to the year I worked as a house officer at the Yorkhill site of the Royal Hospital for Sick Children in Glasgow, Scotland and to memories of children with acute rheumatic fever and others with acute glomerulonephritis. These diseases occur much less frequently now, at least in Western Societies.

My most striking memories of tuberculosis patients in South Africa center on young children with tubercular meningitis. I have a distinct memory of a young child lying quite still in a cot whimpering rather than crying and only partly conscious. This child subsequently died and I attended the autopsy. The brain was covered in a thick greenish colored slime, likely composed of bacteria, white and red blood cells and degenerated tissue. It was this that deprived the child of life.

In 1963, when I was a final year medical student on rotation in the Pediatric inpatient service in the Academic Hospital in Pretoria, South Africa, the senior physician instructed me to enter very quietly into a room to observe a single patient there. He gave further instructions; there was to be no talking, no swift movements and the patient should not be touched. On entering the dimly lit room I observed a single tiny infant lying in a crib. The infant’s hands were tightly clenched, his whole body was tensed and his back was slightly arched. The face muscles were tensed as though the infant was crying but he emitted no sounds. The senior physician then signaled to me to exit the room. He later explained that this was a case of neonatal tetanus. The patient had been brought in from a rural district and the physician suspected that the umbilical cord stump had likely been smeared with animal products, possibly with animal dung.

The following sections are included:

• Hepatitis Viruses and Hepatoma
• Discovery of Hepatitis B Virus
• Hepatitis B Virus and Hepatoma
• Development of an HBV Vaccine
• Successes Achieved with HBV Vaccine
• Hepatitis B Worldwide
• Hepatitis B and Hepatitis C in Hepatocellular Carcinoma
• Hepatocellular Carcinoma and Hepatitis B and C Viruses Worldwide
• Attempts to Control Hepatitis C Virus Infections
• Mycotoxins
• Human Papilloma Virus, Cervical and other Cancers
• Development of a Vaccine that Induces Immunity to Human Papilloma Viruses
• Herpes 8 Virus and Kaposi Sarcoma

My own experience of the impact of HIV/AIDS is South Africa related to children. In 2008, I visited an orphanage for infants and children whose mothers were severely affected by AIDS and were unable to take care of them. No other family members had come forward to care for the children. Some of the orphans were also positive for HIV and were receiving treatment. On another occasion, while on a visit to the Chris Hani hospital in Soweto near Johannesburg, I was taken to a clinic for Pediatric HIV/AIDS patients; many of the children were very thin and seemed much debilitated. I noticed that many children were brought to the clinic by grandmothers and was told that many of the children lived with their grandparents, since parents were not available to take care of them.

In recent years, and particularly in 2015, there have been many reports on antibiotic resistant micro-organisms. In 2015, several national and international incentives were established to promote discovery of new antibiotics to add to the clinical pipeline. In 2016, Brown and Wright noted: “Lessons from the history of antibiotic discovery and fresh understanding of antibiotic action and the cell biology of microorganisms have the potential to deliver twenty-first century medicines that are able to control infection in the resistance era.”

Blair et al. (2015) reviewed mechanisms of antibiotic resistance. In the introduction to their review they noted that each year approximately 24,000 people in Europe die as a result of multi-drug resistant infections. In the USA, approximately 23,000 deaths per year are attributed to antibiotic resistance.

Jensen et al. (2014) reviewed aspects of genomic-based natural product research. They emphasized that genomic sequencing provides a means to assess the biosynthetic potential of micro-organism strains. These assessments include possibilities of strains to produce specific compounds, e.g. polyketides and terpenes. Jensen et al. noted that bioinformatics expertise and lack of information on pathways has hampered progress. They noted further that highly repetitive sequence motifs occur in association with biosynthetic genes and that these impair sequence assembly. They noted further that even when sequencing reveals that specific genes are present that potentially encode enzymes in specific biochemical pathways, the products of these enzymes are not always present or are not identified.

The Human Microbiome Project was designed to characterize the microbiome at different sites in the body and to determine the microbiome status in health and disease. Initial studies involved analyses of the body sites in 300 healthy individuals. In a review of initial results, Ding and Schloss (2014) reported that microorganism taxonomies of the mouth and gut were different but related. They also reported that the microbiome showed considerable intra- and inter-personal variations. They also reported that the male and female microbiomes differed. The gut microbiome enterotypes were found to differ dependent on the content of fat and carbohydrate in the diet. Clear differences were demonstrated in the microbiome of breast fed infants and non-breast fed infants.

Studies on bacterial microfilms have provided new information on microorganism behavior and yielded insights into infections. Biofilms form on biological and non-biological surfaces. The latter include catheters, medical devices, intubation tubes, prostheses and medical equipment. Biofilms are a particular problem in the healthcare setting. Kostakioti et al. (2013) reviewed aspects of biofilm generation. They noted that developmental changes occur in the organisms that form biofilms. The key developmental changes involve transformation from planktonic bacteria that float in liquid medium to bacteria that are anchored in an extra-cellular matrix that they produce. The matrix that bacteria produce protects them from host immunological processes. In addition, inter-bacterial interactions occur within the matrix and facilitate the spread of drug resistance.

In 2014, the World Health Organization urged countries to develop and finance plans to address the antibiotic resistance problem. In the USA, Center for Disease Control (CDC) formulated the National Strategy for Combating Antibiotic Resistant Bacteria (http://www.cdc.gov/drugresitance/threat-report-2013). There are five key goals of this strategy. The first is to reduce emergence of resistant bacteria and spread of resistant infections through more judicious use of antibiotics in healthcare systems and in the agricultural industry.

Reliable of point of care diagnostic assays that facilitate diagnosis of the microorganisms responsible for disease in a specific patient and that provide information on the antibiotic sensitivity of the responsible organism are urgently needed.

There are ongoing efforts to develop tests that measure biomarkers in patients that help to distinguish viral infections from bacterial infection. The hope is that such tests will avoid the inappropriate use of antibiotics in patients with viral diseases. One such test involves measurement of the protein procalcitonin (PCT). Pfister et al. (2014) reported that procalcitonin is a reasonably accurate marker for detection of bacterial pneumonia in critically ill patients. Tsalik et al. (2016) reported that that presence of raised levels of procalcitonin in blood was 78% accurate in predicting bacterial infections in the acute care setting. These investigators developed a test that measured a panel of biomarkers based on altered host gene expression that achieved 87% accuracy in distinguishing between bacterial and viral infections.

In the 21st century, there is marked rejuvenation of vaccine research and evidence of increased vaccine use. These changes are simulated in part by the increasing frequency of microorganism resistance to antibiotics and in part by the increasing burden of viral diseases against which effective antibiotics have not been developed.

Barocchi and Rappuoli (2015) discussed new approaches and models to fund development of vaccines and other medications needed in low income countries. The Advance Market Commitment (AMC) established by the Global Vaccine Action Plan (GAVI) promoted donors to subsidize the costs of vaccines that are needed in developing countries. AMC also functions in promoting suppliers to make funds available for research development and staff training.

My introduction to studies in genetics began when, in 1966, I undertook a research project to study sex chromatin in a series of patients. The study specifically involved studies of the so-called Barr bodies in epidermal cells in buccal smears and the study of specific appendages, known as drumsticks on polymorphonuclear white blood cells present in blood smears.

The following sections are included:

• Adventures in Biochemical Genetics
• Testing Hypotheses on the Subunit Structure of the ADH1, ADH2 and ADH3 Gene Products
• Molecular Genetic Studies on Alcohol Dehydrogenase
• Additional ADH Gene Loci
• Molecular Analyses of Individual Variation of ADH Genes
• Use of Polymerase Chain Reactions (PCR) to Reveal ADH Polymorphisms
• Class I ADH Genes and Population Studies
• Clinical Relevance of ADH Studies
• Fetal Alcohol Exposure

Following the period in 1965–1966 when I undertook studies concentrating on nuclei, chromatin and chromosomes in human cells and took courses in biochemistry and plant genetics, I mentioned my interest in carrying out work in human genetics to a faculty member known for his work on bacteriophages. He dismissed my interest with the comment: “You cannot do genetics in humans, their generation time is too long”. This put-down comment was in fact inaccurate.

When I joined the faculty at the University of California, Irvine in 1981 and had the opportunity to collaborate with faculty and students in a molecular genetics unit within the Department of Microbiology in the Medical School, I realized that it was time to catch up on development in molecular genetics. I spent time reviewing the history and reading the literature.

In 1988, the National Research Council (NRC) in the USA recommended “mounting a special effort to map and sequence the human genome”. They recommended development of maps of polymorphic markers and maps of coding DNA segments in initial efforts (McKusick, 2006). They also recommended emphasis on technological developments related to mapping and sequencing. The NRC proposed support of medium-sized multi-disciplinary centers. In such centers, they suggested that interactions between biologists, physicists, engineers and information scientists should take place.

“The uniformity of the earth’s life, more astonishing than its diversity, is accountable by the high probability that we derived from a single cell……”.

In this section I will review evidence that has come to light on the role of specific founder mutations that occur with high frequency in certain populations and likely account for the fact that I encountered more patents with specific disorders in some locations where I worked.

In the early 1960s in South Africa, while I participated in collecting blood samples on patients who had had heart attacks to monitor their blood levels of the anticoagulant medication (warfarin) they were prescribed, important information relevant to the cause of coronary heart disease was being gathered in the USA. In 1961, evidence gathered from the Framingham heart study in the USA revealed that abnormal cholesterol levels and high blood pressure were associated with heart disease (www.framinghamheartstudy.org/about-fhs/research-milestones.php).

In recent decades, studies in South Africa have revealed evidence that heart disease associated with cardiomyopathy (disorders involving the heart muscle) was due to specific mutations that occurred with higher frequency in individuals with the Afrikaner (Dutch) heritage.

As medical students in South Africa, we were taught that Porphyria was a disorder to be considered in the differential diagnosis of adult patients admitted as surgical emergency cases. A professor of surgery reminded us that if a patient presented with abdominal pain, and on examination was found to have multiple abdominal surgical scars from exploratory surgery, we should consider a diagnosis of porphyria and we should order the appropriate clinical chemistry tests. In addition, we were taught that porphyria patients could present with neurological, psychiatric or cutaneous symptoms.

During my time at Mount Sinai School of Medicine, I also benefited from interactions with clinicians and scientists focused on patient care and research related to Gaucher disease type 1. This disease is also known as non-neuronopathic Gaucher disease. It is an autosomal recessive lysosomal storage disease and affected individuals are most commonly homozygous for a specific mutation. Type 1 Gaucher disease occurs in many different populations, however it is more common in the Ashkenazi Jewish population than in other populations. This disease is due to defects in the function of the enzyme beta glucocerebrosidase (GBA).

During our final year of medical school in 1963 a pediatrician presented the case of a young girl who had severe anemia, enlargement of the spleen and thickening of bones of the skull and face. She required frequent blood transfusions. The pediatrician noted that her condition was likely due to impaired regulation of synthesis of hemoglobin. This statement was intriguing but the underlying mechanism was unclear.

The most memorable patient I encountered during the short period in 1966–1967 when I served as a house officer in the medical team at the Children’s Hospital in Yorkhill, Glasgow was an approximately three-year-old boy, whom I will call Charlie.

As a house officer involved in the care of newborns at the Queen Mother’s Hospital in Glasgow, Scotland in 1967, one of my responsibilities was to work along with the nursing staff to check records and documentation that each newborn had, within the first 48 hours post-partum, satisfactorily passed meconium, the first stool. Meconium contains mucus, degraded cellular materials and bile. Failure to pass meconium could indicate intestinal obstruction in the infant. One form of intestinal obstruction was referred to as meconium ileus and it could be an early manifestation of cystic fibrosis.

Courses and seminars in biochemistry and metabolism during my student years and later clinical responsibilities led to my education and growing interest in inborn errors of metabolism. Perhaps the most important aspect of these activities was the realization that correct diagnosis of a specific inborn error of metabolism could, in some cases, lead to therapy that would improve the lives of patients.

The following sections are included:

• Therapies in Modern Times
• Addressing Root Causes
• Complex Causation
• Genome-wide Association Studies (GWAS)
• Gene–Environment Interactions and Natural Selection
• The Move Toward Personalized Precision Medicine

My interest in Human Genetics was initially stimulated during psychiatry rotation as a medical student and I have retained this interest throughout my career. I continue to be inspired by the modern emphasis on psychiatric disorders as brain disorders that are also impacted by environmental and social conditions.

In an outstanding review of aging in 2013, Lopez-Otin et al. documented factors that play roles in aging and noted biological hallmarks of aging. These included genomic instability, telomere loss, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion and impaired intercellular communication.

A number of different age-related neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease, amyotrophic lateral sclerosis (ALS, Lou Gehrig’s disease) and frontotemporal dementia, have certain histopathological and biological features in common. These include the accumulation of protein aggregates, protein misfolding and perturbations in the processes by which misfolded proteins are eliminated. In addition, there is evidence of impaired trafficking between cellular organelles and disturbed organelle function. Particularly important are defects in functioning of endosomes and lysosomes that are involved in taking up damaged proteins, and the structure and impaired function of mitochondria (Tofaris and Schapira, 2015). Together these impairments lead to degeneration of neurons. Another feature common to different neurodegenerative diseases is the accumulation of abnormal forms of the tau protein.

In the future we can anticipate that pluripotent stem cells and differentiated cells derived from stem cells will increasingly be utilized to analyze the downstream effects of gene mutations and to develop and to test novel therapies.

This refers to the modification of specific genome sequence targets through the use of nucleases, enzymes that cut DNA. Gene editing can be used as a method to modify genes in in vitro conditions, to assess the functions of specific genes and to assess the downstream effects of specific gene mutations.

It is likely that in the next years there will be progress in identifying specific nucleotide mutations that lead to particular genetic diseases. It is also likely that therapies will be designed to neutralize the effects of these mutations. Antisense oligonucleotides represent one form of therapy that will be applied.

Extensive studies are ongoing to analyze tumor DNA through next generation sequencing and to match mutations with specific therapies that target tumor-specific mutations. One such program is the USA National Cancer Institute (NCI) trial designated MATCH (Molecular analysis for therapy choice). This trial currently specifically enrolls patients with advanced solid tumors or lymphomas that are not responding to standard therapies. The goal is to search for genetic mutations that may respond to specific target therapies. In patients recruited to the MATCH trial, mutation testing and drug costs are covered by the NCI (https://www.cancer.gov/about-cancer/ treatment/clinical-trials/nci-supported/nci-match).

In many countries in the world, health and well-being have increased significantly over the past several decades. Are these improvements due to improved living conditions or due to improved healthcare? The answer I believe is that both are important.

The following sections are included:

• Expanding Concepts of Health and of the Dimensions of Environmental Impact
• Planetary Health
• ARBO viruses
• Diseases Caused by ARBO Viruses
• Zika ARBO virus
• Convergence
• References

The following section is included:

• Index