Chemokines and Stealth Viruses:A Blueprint for Therapy in Infected Humans and Animals

© Copyright 2002 by W. John Martin, M.D., Ph.D., USA
(Explore Issue: Volume 11, Number 1)

Chemokines

Relatively small proteins (peptides) that mediate intercellular signaling among lymphocytes responding to antigenic challenge, were originally referred to as lymphokines. This term was replaced by interleukins, since lymphokine did not fully reflect intercellular communications between lymphocytes and cells of macrophage/monocyte lineage. It was further realized that related, and sometimes even identical peptides, were being produced by and exerted growth regulatory effects on cells outside of the immune system. The more inclusive term cytokine was, therefore, applied for locally acting peptide molecules that provided intercellular signaling for multiple cellular systems. Several cytokines were named for the cellular modulating property with which they were initially associated. For example, tumor necrosis factor (TNF) was shown to cause death of certain tumor cells; melanoma growth stimulatory activity (MGSA) was named for its ability to stimulate melanoma cells, etc. Other cytokines were defined by their cellular location, for example nuclear factor-kappa B (NF-kB).

A subset of cytokines was found to have an additional property of mobilizing and attracting cells towards their source. These chemo attractant cytokines were termed chemokines. An interesting structural correlation has emerged regarding the presence of cystine disulphide bonds within chemokine molecules, and the types of immune cells bearing receptors able to receive the migratory signal from the chemokine. Specifically, the beta class of chemokine, which primarily attracts monocytes, possesses two adjacent cysteine amino acids within the left side of the molecule. These cysteines interconnect with two distally placed cysteines to form two disulphide cystine to cystine bonds. Beta chemokines are designated CC because of the adjacent positioning of the pair of cysteine amino acids. Alpha chemokines, which primarily attract neutrophils, and to some extent lymphocytes, have a single amino acid separating the pair of cysteines, and are designated CXC chemokines. MGSA is an example of a CXC chemokine, as is interleukin-8. Fractalkines are primarily involved in neuron-glial cell interactions, but can also influence immune function. They have three amino acids separating the cysteine pair, and are designated CX3C. Lymphotaxins are a fourth class of chemokines with a single disulphide bond and are designated C. Multiple representatives of CC and CXC chemokines have been described, in comparison with only a few examples of CX3C and C chemokines.

 

A corresponding diverse array of chemokine receptors has been identified. Some are highly specific for limited members within a chemokine class, while others can allow binding of multiple chemokines, even belonging to different classes. The CC, CXC and CX3C receptors are designated numerically in the order that they were identified, for example CXCR4 was the fourth type of receptor identified that preferentially binds a CXC chemokine. The receptors are cell surface proteins with seven segments that traverse the cell membrane and an intracytoplasmic tail that interconnects with a signal transducing group of proteins known as G proteins. Receptor binding can lead to a series of changes within the cell, many of which are mediated by the transfer of a phosphate residue from cyclic guanosine triphosphate (GTP).

Viruses and Chemokine Receptors

Scientific interest in chemokine receptors was boosted with the realization that human immunodeficiency virus (HIV) isolates could utilize the CXCR4 and /or CCR5 chemokine receptors in order to gain entry into lymphocytes. Indeed individuals inheriting certain genetic forms of these receptors were far less prone to HIV infection than was the general population. Moreover, in lymphocyte cultures, occupancy of the receptors with their corresponding chemokine, could inhibit viral infection. Chemokine receptors are used by several other viruses, many of which also code for chemokines and/or chemokine receptors. Human cytomegalovirus (HCMV) contains a gene that codes a potent CC chemokine receptor. The gene is designated US28 since it is the 28th gene along a unique short segment of the HCMV genome. The other segment is called the unique long (UL) segment and codes up to 154 genes. The US28 gene of HCMV can provide intracellular access for HIV and can also act as a trap to diminish the probability that CC chemokines would be blocking the cellular receptors on other T lymphocytes. CC chemokine receptors are also coded by other herpes viruses, including human herpesvirus-6 and human herpesvirus-8. HCMV and mouse cytomegalovirus also code for CXC chemokines and can induce cellular chemokine production from infected cells. The tissue culture infectivity of HCMV is enhanced in the presence of interleukin-8, a CXC chemokine.

Stealth Adapted Viruses

In a process termed stealth adaptation, viruses can avoid elimination by cellular immunity, if they lack genes coding the relatively few structural components that are targeted by cytotoxic T lymphocytes (CTL). An atypical African green monkey simian cytomegalovirus (SCMV) was isolated from a patient with the chronic fatigue syndrome (CFS). It caused no inflammatory reaction within the patient, nor when inoculated into cats. The cats did, however, develop widespread signs of cellular damage including the development of foamy, vacuolated cells throughout the brain. Similar changes were readily induced in cultures of human and animal cells. Since isolating this virus, evidence for stealth virus infection has been found in numerous patients with a wide spectrum of neuropsychiatric and immunological diseases. The diagnoses applied to stealth virus infected patients have varied depending upon the major clinical manifestations, and in part, upon the clinical background and perspective of the diagnosing clinician. The illnesses have included autism, behavioral and learning problems in children; depression, chronic fatigue, fibromyalgia, Gulf war syndrome, multiple sclerosis and severe psychosis in adults; and degenerative neurological diseases, including Alzheimer's, Parkinson's and amyotrophic lateral sclerosis, in the elderly. The viruses found in various patients with these diseases were termed stealth because of their apparent inability to evoke an inflammatory response and partly because they had gone undetected by earlier investigators seeking an infectious cause for such illnesses.

DNA sequencing studies on the prototype stealth virus confirmed the deletion of the gene coding the dominant antigen (UL83) recognized by anti-cytomegalovirus CTL. Deletions and major disruptions were also present in several of the other genes known to code significant antigens normally targeted by anti-cytomegalovirus CTL. The question arose as to how such a virus could retain and/or regain its ability to damage cells. A series of experiments showed that the virus had a fragmented, genetically unstable genome. Moreover, it had assimilated various additional genes both from infected cells and also from various bacteria. More recent studies have implicated chemokines and chemokine receptors in the biology of this stealth adapted virus.

Melanoma Growth Stimulatory Activity (MGSA) Chemokine Coding Genes

Sequencing of cloned fragments obtained by restriction enzyme cutting of the SCMV-derived prototype stealth virus has revealed complex patterns of viral, cellular and bacterial sequences. One region of the virus shows a linear alignment of the following genes UL141, UL144, UL145, an undefined region of probable cellular origin and three genes most closely related, in terms of protein coding, to the cellular alpha chemokine gene, MGSA. The stealth viral genes differ somewhat from each other but have all retained the defining CXC arrangement of the cysteine amino acids. The genes were assimilated from an RNA molecule since they generally lack the non-coding intron components that are the parts of cellular DNA that are excluded from cytoplasmic RNA. This finding indicates that stealth virus replication has involved reverse transcription, presumably by a cellular homologue of a retrovirus. While MGSA is now regarded as a prominent alpha-chemokine, it is also considered a potential cancer-causing gene (oncogene). The finding of this gene has raised the prospect that stealth adapted viruses may carry other oncogenes and be responsible for the significant increases in various types of cancers. Indeed, in early studies many cancer patients are testing positive for stealth viruses, in contrast to healthy blood donor. The prospect of oncogenic stealth-adapted viruses evolving from replicating viruses through the assimilation of an oncogene provides an imperative to hasten the development of at least suppressive, anti-stealth virus therapies.

Chemokine-Receptor Coding Genes

Continued sequencing of the prototype stealth adapted virus identified another segment in which the following genes were present: US24, shortened US26 and five genes related to US28 chemokine receptors. The five genes have diverged somewhat from each other yet have retained critical amino acids in common with the US28 gene of HCMV and the CX3CR receptor which is the human cellular gene that most closely matches the stealth virus genes. The next best matching set of cellular genes was CC receptors, with one of the five genes also showing a good match to a CXC receptor.

Based on the demonstration that the prototype stealth adapted virus carried expanded copies of both chemokine and chemokine receptor coding genes, it seemed likely that, at least this virus, was been driven and/or was exerting some of its pathological effects through chemokine mediated pathways of cellular activation.

Chemokines and Their Receptors as Targets for Therapy

The initial association of chemokines as mediators of immune responses stimulated the identification of drugs that could affect chemokine mediated pathways of lymphocyte activation. Immune enhancement was a major goal of potential cancer therapies, while immunesuppression was desired for patients with autoimmune diseases and for transplant recipients. This endeavor expanded considerably with the realization that chemokine receptors were involved in HIV infection. These developments are particularly timely in view of the data obtained on the prototype stealth virus.

During the last several years, useful information has been obtained regarding various therapies that appear to have offered benefit to stealth virus infected patients. Cultures have been noted to have gone from rapid strong positive to delayed weak positive after the institution of various empirical therapies. Several of these approaches have involved what is generally regarded as alternative medicine. It is striking that many of these therapies have been linked to possible inhibition of cytokine/chemokine production and activity. Up to the present, however, there has been little guidance on which of the many treatment options available should be pursued and how, apart from the subjective feeling of wellness, to regulate dosage. While individual chemokines can be measured, there is insufficient data to relate these levels to stealth viral load. The availability of a semi-quantitative assay of stealth virus infection provides a more direct laboratory reflection on treatment efficacy.

Partial Listing of Agents Known to Influence Chemokine Production and Activities

The accompanying table is intended to illustrate the vast number of agents that have been reported to influence the production and/or activities of CC and CXC chemokines. The cytokine NF-kB is a potent stimulus to chemokine production. NF-kB is normally maintained in an inactive state due to inhibitory molecules. Activation can occur due to the production of TNF and other cytokines. In turn, TNF production can be evoked by various factors, including oxidative stresses. Chemokine production can be regulated, therefore, by agents that act at various levels, including suppressing the production of oxygen free radicals with antioxidants, suppressing TNF and/or NF-kB production, directly acting on chemokine production or interfering with the production and/or activity of chemokine receptors. Activation of certain other pathways can lead to inhibition of chemokine production, for example prostaglandin E (2), interleukin 10 and their inducing agents can down regulate chemokine synthesis. Several compounds can have opposing actions, depending on dose, cell type and inducing stimulus.

-As shown in the above table, a surprisingly large number of dietary supplements, herbal medicines, antibiotics and other commonly used drugs, have been shown to have an influence on chemokine production and activity. The list is incomplete and many more compounds may, on the appropriate testing, be shown to down regulate certain chemokines.

Useful information for the ultimate development of a standardized protocol to assist stealth virus infected patients should be forthcoming from serial determinations of levels of stealth virus infections on blood samples obtained prior to and following 2-4 week courses of various agents such as those included in the above table. For patients with minimal illnesses and especially in children, the initial emphasis should be on non-toxic dietary supplements and on over-the-counter medications. Thus it can be argued that maximum benefit from relatively non-toxic medications should precede the use of potentially toxic agents. If simple therapies fail, or if faced with more serious illnesses, it is appropriate to proceed with therapies that require professional medical dispensing and oversight. These drugs include a host of commonly prescribed anti-rheumatic medications, certain antibiotics and other drugs not widely known to influence chemokine levels and/or activity.

The clinical benefits seen with antibiotics have been ascribed to their ability to destroy pathogenic bacteria. The postulated targets for activity have included Borrelia (the cause of acute and classical Lyme disease), mycoplasma (a postulated cause of Gulf war syndrome and CFS). Chlamydia and rickettsiae (also presumed by some to be involved in the pathogenesis of CFS). As argued elsewhere, many of the assays supposedly supporting the presence of such bacteria, may actually reflect the assimilation of various bacterial sequences into stealth adapted viruses. The term "viteria" has been applied to stealth viruses that have acquired bacterial sequences. The pattern of clinical response to antibiotics is as consistent with an anti-chemokine action as with eradication of pathogenic bacteria. Furthermore, the use of antibiotics has the potential adverse effect of altering the body's bacterial flora. For this reason, it is probably preferable to try herbal and anti-rheumatic drugs before resorting to antibiotics. An exception to this argument is when specific testing identifies the presence of atypical bacteria or fungi that can be shown to be viteria infected.

The list does not include the use of accepted anti-viral agents, such as ganciclovir. Nor does it include therapies that are designed to address symptoms resulting from viral induced organ damage, especially damage that has occurred to the brain, endocrine organs, gastrointestinal tract, heart, blood, coagulation system, etc. Therapies for these and other manifestations of stealth viral illnesses need to be addressed on an individual basis.

Summary

While stealth virus research is still in the phase of discovery and characterization of individual isolates, it is important that efforts be underway to suppress the level of infection in those already infected. This may help minimize the risks of viral transmission and may also reduce the chances for emergence of more virulent, and even potentially oncogenic, stealth viruses. The present review has focused on agents that can influence cytokine/chemokine levels. This approach is bolstered by what is known about chemokines and chemokine receptors in conventional herpes viruses, data indicating expansions in the number of these genes in a prototype stealth adapted virus and by the knowledge base that many of these approaches have provided some relief to patients with diseases potentially due to stealth virus infections. Stealth virus monitoring of patients undergoing various therapies should help expedite the optimization of treatment protocals. Additional information and copies of published articles on stealth viruses are available from the web site www.ccid.org

Articles by Dr. W. John Martin on his stealth virus research can be found in volume 10, 4 page 17 as well as Volume 10, 5 page 7 of Explore. We are grateful to Dr. Martin for sharing this valuable research with our readers and thank You, the reader for the many positive responses. Please contact Dr. Martin directly at Center for Complex Infectious Diseases (CCID) at 3328 Stevens Avenue, Rosemead CA 91770, (telephone (626) 572-7288, Fax (626) 572-9288, e-mail ccidlab@hotmail.com) as we are not knowledgable in this field to answer Your questions. Thank You.

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