Tuesday, October 29, 2019

Microbiota and Immunity in Healthy Human Organisms

As a result of the millions of years of the evolution of the human species, the human body has established a commensal association with microbiota (microorganisms). This mutually beneficial relationship is essential for the maintenance of the health of the human host. However, it appears that when the natural barriers are disrupted, disease can result. The normal balance (homeostasis) between microbiota and host is apparently maintained by a robust immune system. The nature of this natural process, however, is poorly understood.

The current evidence is that specialized immunocompetent cells are implicated in this process. These are referred to as mucosal-associated invariant T (MAIT) cells and that these MAIT cells recognize and react to metabolites that are that are a byproduct of microbial metabolism. These metabolites are believed to play a role in microbial defense.

Drs Constantinides Legoux and their colleagues have reported that commensal bacteria exert control of the development of MAIT cells in the thymus (the organ involved in the development and proliferation of T cells) and their subsequent expansion within mucosal tissue. Additionally, the development of MAIT cells depends upon exposure in early life to well-defined microbial communities. Furthermore, it appears that a distinct subset of MAIT cells is actively involved in wound healing.

What makes MAIT cells distinct is the fact that in their activity they do not recognize the major histocompatibility complex (MHC) molecules like classic T cells. Instead, they are stimulated by nonpeptide (non-protein) antigens such as vitamin B2 precursor derivatives that are produced by many bacteria bound to an MHC-like protein referred to MR1.C-like H

Several recent studies point to an even broader range of activity for MAIT cells including: the control of bacterial, fungal and viral infections, a role in autoimmune disease and possible involvement in the immune processes involved in attacking the proliferation of tumor cells.

The results of extensive study of MAIT cells coming from the laboratories of Constantinides and Legoux have established that MAIT cells require a community of microbiota to enable their development in the thymus and insure their expansion into specific tissues. It seems that, in fact, newborns are rapidly colonized by a diverse complement of species and strains demonstrating the key role that these microorganisms play in establishing the healthy status of the rapidly developing new human. Furthermore, in humans, it appears that the frequency and localization of MAIT cells throughout the organism changes over a lifetime and is apparently diminished in the elderly population.

In summary, the diagram below illustrates the intimate relationship that exists between the development and function of MAIT cells and the resident commensal microbiota on barrier tissues such as the skin. These finding also emphasize the key role that these organisms play in the health of individuals beginning at birth and proceeding throughout an individual lifetime.

Sunday, August 11, 2019

Cancers Caused by Contagious Agents

We do not generally regard cancer as being contagious. Although this is generally t rue, there are a growing number of cancers that have been shown to be caused by contagious agents, especially by specific viruses. The types of cancers caused by such agents are shown along with the microorganism implicated.

Cancer Causative Agent

Stomach (Gastric) Cancer H. Pylori - Bacterium
Cervical Cancer Human Papilloma Virus (HPV)
T-Cell Leukemia HTLV-1 – related to the AIDS virus
Burkitt’s Lymphoma Epstein Barr Virus (EBV)
Kaposi’s Sarcoma HTLV-3 – AIDS Virus
Primary Liver Cancer Hepatitis B Virus (HBV)

In regards to the data listed in the table above, there is additional information associated with them:

• H.Pylori is a highly specialized and fairly ubiquitous bacterium that can survive in the harsh acidic environment of the stomach; it is found in the mucus layer on the inside of the stomach within those individuals who are infected. For this reason, it is particularly difficult to treat with antibiotics. It is found in about 2/3 of the world’s population. It has also been implicated in the onset of chronic gastritis and peptic ulcers.
• HPV has recently been implicated as the causative agent for cervical cancer. Cervical cancer is prevalent in sexually active women. A vaccine against this virus has recently been developed. It has been shown to be highly effective in preventing the onset of this cancer in young women.
• EBV, the virus that causes Burkitt’s lymphoma found predominantly in Africa, is the same virus that causes mononucleosis in individuals in the West. The reason for this distinct difference in disease outcomes is poorly understood, but a genetic basis for this difference is likely.
• The AIDS virus is not directly responsible for the onset of Kaposi’s sarcoma. The disease is manifested in AIDS patients on account of their highly suppressed immune system due to infection with the AIDS virus that make AIDS patients especially susceptible to this cancer.
• Once an individual is infected with HBV as a result of close human contact with an infected individual, the virus can infect the liver without any noticeable symptoms. This can be the case for many years before the onset of liver cancer. HBV is believed to account for 80% of the reported cases of primary liver cancer (cancer that originates in the liver). It is a deadly cancer. Fortunately, there is a vaccine against HBV infection, but there is currently no vaccine to prevent infection by the Hepatitis C Virus (HCV) that is also known to cause primary liver cancer.

Control of Inflammation following Injury or Infection

The natural immune responses that are elicited after an insult to the body as a result of injury or infection from a pathogen, is both rapid and powerful. Initially inflammatory cells are mobilized to the site of the trauma. The predominant cell types that are recruited are macrophages and neutrophils that release free radical reactive oxygen and nitrogen species (RONS) that are lethal to invading organisms. However, these chemical moieties are so powerful that they can also kill and mutate the surrounding normal tissue. Although there are naturally induced anti-inflammatory responses that are also initiated, the optimum balance is difficult to achieve. In some diseases, such as ulcerative colitis and rheumatoid arthritis, such anti-inflammatory responses are unavailable. Therefore, for this reason medical intervention in the form of medication becomes appropriate. 

There are, of course, many different anti-inflammatory medications currently available. Recently Dr. Torkild Visnes and his colleagues from the Karolinska Institutet in Sweden in collaboration with the University of Texas Medical Branch, Uppsala University and Stockholm University have discovered a new methodology for enhancing the anti-inflammatory response. In order to understand the investigator’s approach, we need to examine the rationale for this research in greater detail. 

Normally the enzyme 8-oxyguanine DNA glycosylase 1 (OGG1) functions as a DNA repair enzyme that both recognizes and repairs the nucleotide base excision repair of 7,8 dihydro-8-oxoguanine (8-oxoG) that represents one of the major types of DNA damage produced by RONS. Paradoxically, the binding of OGG1 to 8-oxoG, facilitates the action of the NF-kB transcription factor that promotes the activation of quiescent chemokine and cytokine genes that subsequently leads to the inflammatory response and the subsequent release of RONS at the site of trauma. 

Visnes and his group have identified a small molecule (TH5487) that binds to the active site of OGG1 and effectively blocks its repair capabilities on account of the fact that inhibited OGG1 cannot bind to that G-rich region of the DNA leads to the activation of the NF-kB transcription factor that promotes proinflammatory genes. In fact, TH5487 has been shown to inhibit this process in mouse and human lung epithelial cells in vitro and the TNF-induced neutrophil inflammation in the in vivo mouse model. 

This research is of value since it elucidates a molecular mechanism that demonstrates a connection between the normal DNA repair function of OGG1 and the inflammatory response and has discovered a small molecule inhibitor of this process.

Saturday, August 10, 2019

A Study in the Evolution of a Transmissible Cancer

There is a canine-related cancer that is sexually transmitted from one animal to another. It is the canine transmissible venereal tumor (CTVT) that presents as genital tumors. It has been shown that this cancer spreads through the transfer of living cancer cells (see image below) through coitus. These CTVT cells function as a unicellular, asexually reproducing (but sexually transmitted) pathogen. In addition, CTVT cells are genetically aberrant in terms of chromosome number – studies have established that instead of the normal 78 chromosomes present in the canine species these cells have a chromosome number in the range of 57-64. This type of cancer represents an exceedingly rare etiology in that the cancer is spread through the transfer of the cancer cell itself.

CTVT Cells
This disease exists worldwide and apparently has the longest known and most prolific evolutionary lineage. This reality provides an opportunity to explore the mechanism of the evolution of cancer over the long term. For this reason, Adrian Baez Ortega, a bioinformatician, and her colleagues from the Transmissible Cancer Group at Cambridge University, UK, have done a detailed analysis of genetic sequence data from 546 individual CTVT tumors taken from diseased animals. Their focus in this analysis was to identify somatic single-nucleotide variants (SNVs) from the exomes – areas of the genetic material that are involved in the active production of proteins. The aim of this intense research study was to help to uncover the mutational events and genetic signatures of the evolutionary selection process over the thousands of years.

One of the predominant findings of this analysis was that this lineage arose from its originator canine individual between 4000 and 8500 years ago most likely from Asia with the most recent ancestor of the current global distribution estimated to have lived about 1900 years ago. There does not appear to be any positive selection for this lineage. In addition, the author of this study concludes that, “a highly context-specific mutational pattern named signature A was identified, which was active in the past but ceased to operate about 1000 years ago. BP, years before present.”

These results provide an insight into the progression of an unusual type of cancer that is transmissible through the transfer of the cancer cell itself rather than through a vector such as a virus – human T Cell Leukemia being one particular example. It also is a demonstration of the power of the widely used tools of research regarding the discovery of the underlying genetic mechanisms involved in disease processes.

Friday, June 7, 2019

Senescent Cells and Human Longevity

Although the average human lifespan has increased substantially over time due to the improvement in living conditions made possible by advances in public health, sanitation, medicine, etc., there is no selective advantage afforded by a longer life once the reproductive period has passed. Consequently, there is a normal and gradual deterioration of the tissues that is age-related.

Furthermore, there is a natural process referred to as cellular senescence – cells undergoing this change are no longer able to divide. Cellular senescence confers a reproductive advantage for the individual in that it helps block cancer cell proliferation; however, overtime it results in an increasing abundance of senescent cells (SNC) within the tissues. It seems that in animal studies, using the mouse model, in which the SNCs are selectively eliminated (senolysis),the median lifespan of individual test mice is extended, and the frequency of age-related diseases has been shown to be diminished. This result has encouraged the search for and development of drugs that selectively target SNCs.

In terms of this research, it is vitally important to discover the actual mechanisms that underly cellular senescence. In studies using cells grown in culture, it has been shown that SNCs are in a state of permanent cell cycle arrest. This state is apparently initiated and maintained by the p53-p21 retinoblastoma (RB) and p16-RB tumor suppressor pathways. The factors that can trigger this process are –
  • Oxidative stress
  • Shortening to telomeres – repetitive DNA sequences at the ends of chromosomes that afford protection
  • Prolonged mitotic activity
  • DNA errors during replication
  • Mitochondrial impairment.
SNCs produce a so-called, “Senescence associated secretory phenotype” (SASP). SASP has been shown to negatively impact normal tissue architecture through a variety of processes including the onset of fibrosis and the inhibition of stem cell functionality. Although SASP has a protective function in regard to the development of cellular neoplasm, in later life it does not seem to provide a protective function against the onset of cancer. This raised the possibility that the selective elimination of SNCs from older patients might exert an anti-cancer effect.

Given this data, it would seem that therapies that can effectively eliminate SNCs might produce a two-fold health advantage by increasing longevity and by decreasing the onset of cancer in later life. Encouraging results from animal model studies have shown that drugs that target those pathways that block apoptosis – programmed cell death – promote senolysis and afford an anti-cancer potential. In regard to future research, this may provide a very fruitful line of enquiry.

Tuesday, February 26, 2019

Microbial Carcinogens in the Human Large Intestine

The microbial micro-environment in the human large intestine is intricate and complex. In fact, there are many hundreds of small molecules and metabolites produced by this diverse population of microflora that may profoundly influence human health. Many of these substances are produced by enzyme-directed pathways that have been shown to be programmed by so-called, “bacterial biosynthetic gene clusters.” 

One class of these molecules, colibactins, has been shown to be produced from a gene cluster called the, polyketide synthase island (PKS). PKS occurs in certain strains of Escherichia coli (E-coli) that seem to be prevalent in the microbiota of colorectal cancer (CRC) patients. Up until this time, despite many years of painstaking research, little has been discovered regarding the structure and the mode of action of colibactins.

Dr. Matthew Wilson and his colleagues at Vertex Pharmaceuticals have recently published a paper in the journal Science in which they describe the mode of action of colibactins. According to the author, “colibactin alkylates DNA in cultured cells and in vivo, forming covalent modifications known as DNA adducts. These colibactin-DNA adducts are chemical evidence of DNA damage and represent a detectable signature of exposure to colibactin. Misrepaired DNA adducts may generate mutations that contribute to colorectal tumorigenesis.”

In their research Wilson’s group identified the colibactin-DNA adducts as involving the cyclopropane ring and that the site of alkylation involves the nucleotide adenine within the DNA backbone (see diagrams below). Furthermore, it is believed that these adducts could lead to mutations in the oncogenes or tumor suppressor genes that drive CRC-related tumorigenesis.

Although, this model of colibactin involvement in DNA modification is significant, many questions remain unanswered in regard to how tumorigenesis is subsequently initiated in CRC. However, it is line of research that offers some promise in elucidating cancer-causing mechanisms.

Thursday, January 24, 2019

Evolution of an Enzyme from Short Peptide Pieces

Enzymes are complex protein molecules that are responsible for accelerating chemical reactions in the living cell that essentially make life possible. Each unique enzyme is responsible for a particular chemical transformation. The sum total of all of these reactions is what is referred to as cellular metabolism. The primary structure of enzymes – the sequence of amino acids embedded with the protein structure – is encoded within the particular gene responsible for the production of each unique enzyme.

It is of great interest to understand how such complex protein structures evolved from simpler structures that are known to have been available early in the evolution of life on planet earth – amino acids and peptide. In regard to proteins, the class of molecules that represent short pieces of protein is referred to as peptides. Peptides are short strands of amino acids tied together through peptide bonds. 

Enzymes generally require complex folding in their structures to foster their catalytic activity. Peptides are generally too short for this folding to occur. Recent research into the structure of metalloenzymes – enzymes that employ metal ions in their structures – suggest that metal ions may have helped induce folding in precursor peptides. Metalloenzymes are ubiquitous in nature and play fundamental roles in cellular biology and chemistry.

Sabine Studer from the Dana-Farber Cancer Institute, Boston and her colleagues have attempted to more fully understand the processes through which enzymes have evolved. They have done so by devising techniques for facilitating the transformation of a peptide capable of binding zinc into a functional enzyme with a complex globular structure.

According to the authors, “Recapitulating such a biogenetic scenario, we have combined design and laboratory evolution to transform a zinc-binding peptide into a globular enzyme capable of accelerating ester cleavage with exacting enantiospecificity and high catalytic efficiency (k cat/K M ∼ 10 6 M -1 s -1). The simultaneous optimization of structure and function in a na├»ve peptide scaffold not only illustrates a plausible enzyme evolutionary pathway from the distant past to the present but also proffers exciting future opportunities for enzyme design and engineering.”

The techniques they successfully employed to accomplish this include (see diagram below)
Computational redesign
Cassette Mutagenesis – An in-vitro technique for altering genetic structure i.e. mutations.
DNA shuffling
Random mutagenesis
Alanine scan – Alanine is one of the amino acids that plays a critical role in protein structure.

Note: that the majority of the techniques employed involve manipulating the genetic information.

The work cited above is of seminal importance in the overall search for elucidating the molecular mechanisms that may account for the evolutionary development of life on planet earth from its elemental beginnings