Not apropos of anything, an ethics question flitted through my mind as I was reviewing a rather interesting paper for a journal, which shall remain nameless. As for all questions of such deep significance and importance, I would love to turn to my most valuable resource, the scientists and/or blogger tweeps with whom I communicate and/or interact and/or whom I follow on Twitter. I do see the social medium of Twitter to be a valuable tool for collaboration, and I hope there’d be someone there, who can answer my question – either in 140 characters on Twitter, or more at length, here in the comments.
Every so often, some paper happens to grab my attention for various reasons. As I read the paper, often I have questions. Not all of those questions, unfortunately, can be easily submitted for answers. In recent times, one such paper was published earlier this month in PLOS One. The great benefit of the Open-Access model of PLOS is that it allows a reader to ask questions directly of the authors. This level of engagement is very laudable, especially to someone like me who has an interest in the communication of scientific facts.
Much to like in a new study published in the journal mBio today. This study (mBio, 2012, 3:e00312-12) by Lin et al.  (led by Brad Spellberg of UCLA) discovered that, for the gram-negative pathogenic bacteria Acinetobacter baumannii, notorious for its association with nosocomial (‘hospital-associated’) infection the world over and highly resistant to antibiotics, shutting down the production of an extracellular antigen released by the bacteria can effectively silence its effects on the host body and enhance its clearance, to the benefit of the host.
Two main reasons why I found this study interesting. First, it falls squarely in line with the hypothesis of Damage Response Framework (DRF) of microbial pathogenesis, put forth by Arturo Casadevall and Liise-anne Pirofski (who is also the editor for the mBio article under discussion), in their seminal Nature Reviews Microbiology paper in 2003  [N.B. COI declaration: I have worked with Liise-anne.] The DRF represents a simple, yet elegant, attempt to synthesize the existing views of infectious disease pathogenesis that are either microbe-centered (i.e. the ability to cause disease – a.k.a. pathogenicity – is an attribute of the microbe, by virtue of its products and/or ability to replicate in the host) or host-centered (i.e. a disease represents a breakdown in the host defence mechanisms, which allows microbes either to cause disease immediately or to colonize niche areas in the host to activate and cause disease later). There are important lacunae in both views taken separately; for instance, a microbe-centric view doesn’t explain occasional pathogenicity of ordinarily benign, commensal organisms such as Candida albicans, while a host-centric view doesn’t explain disease caused by certain microbes in apparently immune-competent individuals. An example of the latter is nosocomial candidiasis associated with intravascular catheters, prostheses and implanted devices.
Dispensing with complicated functional definitions that abound for microbes, the DRF simply defines a ‘pathogen’ as a microbe capable of causing damage to the host, and ‘virulence’ as a relative measure of that capacity, underscoring the concept that “only in a susceptible host is a microorganism a pathogen, and virulence can be expressed.” How does this help the understanding of disease and pathogens? According to the DRF, “…outcome of microbial pathogenesis is the result of a host–microorganism interaction, and that the relevant outcome of this interaction is host damage.” Although the usefulness of the framework may be somewhat restricted until ‘host damage’ is quantified, but for the sake of understanding, a clinically manifest disease may be taken as evidence of damage. Most importantly, the DRF could reclassify common pathogens according to whether or not they could cause disease in presence or absence of a strong or weak host immune response. For instance, the fungal pathogen Aspergillus that causes disease in situations of both weak and strong immunity is in a different class (Class 4) than, say, the bacteria Staphylococcus epidermidis that causes disease only in the setting of weak immunity (Class 1), or the gastric pathogen, Helicobacter pylori, that causes damage if the immune responses to it are strong (Class 6) .
Consider Acinetobacter baumannii, a ubiquitous coccobacillus (shaped intermediate to spherical and rod-shaped) known to be the world’s most prevalent bacterial species present in clinical settings, responsible for a variety of hospital-associated infectious syndromes – many of them life-threatening – such as nosocomial pneumonia (often affecting ICU patients on mechanical ventilation), nosocomial meningitis, sepsis, urinary tract infection, as well as traumatic skin, soft-tissue and bone infection . A. baumannii has the dubious distinction of being one of the few bacterial pathogens that have become resistant to all available FDA-approved antibiotics (‘pan drug resistance’ or PDR), although majority of the hospital isolates are considered extensively drug resistant (XDR) (i.e., resistant to carbapenems and all other antibiotics except colistin or tigecycline) . The unique ability of A. baumannii to survive in a hospital environment and develop resistance has been attributed to several factors: (i) bioﬁlm formation and resistance to dessication on non-living surfaces; (ii) adhesion, colonization and invasion of human epithelial cells; (iii) considerable repertoire of antibiotic resistance mechanisms that can be promptly up-regulated as required; and (iv) acquisition of foreign genetic material via lateral gene transfer to promote its own survival under antibiotic and host selection pressures .
Scanning electron micrograph of a highly magnified cluster of Gram-negative, non-motile Acinetobacter baumannii; Mag-13331x;
Source: CDC’s Public Health Image Library Image #6498; Photo Credit: Janice Carr, a CDC microbiologist and electron microscopist.
So how does A. baumannii cause disease? It appears that this bacteria behaves much like the DRF Class 5 or Class 6 pathogens. In the mammalian host, response to A. baumannii infection is in part hinged on the immune-reactive lipopolysaccharide (LPS) antigen that this bacterium expresses on its cell surface. Bacterial LPS (variants of which are elaborated by A. baumannii and various other bacteria) is a known, potent inducer of inﬂammatory responses, working via the adapter molecule CD14 and the pathogen signature-recognition molecule TLR4 (which I have written about earlier) present on the surface of various immune defence cells of the host. TLR4-dependent LPS signaling processes are involved in release of inflammatory mediators such as Tumor Necrosis Factor (TNF)-α and interleukin (IL)-8 from macrophages (phagocytic cells active in innate immune defence) . Therefore, ordinarily, TLR4-signaling induced by A. baumannii LPS and the subsequent inflammatory response (which calls in reinforcements) are important in protection against the bacterial infection and help clear the microbe from the tissues (via mechanisms such as phagocytosis, in which immune cells internalize and kill the microbe). However, like DRF Class 5 or 6 microbes, A. baumannii has the ability to cause excessive inflammation in the tissues, leading to host damage; for example, in case of ventilator-associated nosocomial infections in immune-competent hosts, the microbe is known to cause severe acute and chronic inflammation of the lung and tissue damage consistent with pneumonia – as also corroborated by animal experiments. At least one recent clinical report has observed a severe inflammatory response, called the Macrophage Activation Syndrome (in which uncontrollably activated macrophages devour other blood cells!), subsequent to A. baumannii infection.
Clearly, a way to control the unmitigated inflammation caused by A. baumannii infection would be beneficial to the host. Conventional wisdom says that reducing the number of bacteria in the tissue by pharmacotherapy, i.e. administration of antibiotics, would also help. This is where A. baumannii throws a spanner into the works by turning XDR, or worse, PDR.
And this is where the study under discussion becomes interesting, by espousing evidence-driven, targeted strategies for therapeutics and drug discovery. The authors had access to XDR A. baumannii strains of two different mouse virulence phenotypes, highly virulent (strain HUMC1) and less virulent (strain ATCC 17989), and to two types of mice, genetically modified or mutated mice that lack TLR4, and their normal ‘wild-type’ counterparts (with a functional TLR4). Surprisingly, the highly virulent strain, that caused wild-type mice to go into septic shock (with attendant hypothermia; acidemia, i.e. lowering of blood pH; higher levels of various cytokines; apoptotic neutrophils in spleen and lung, consistent with Gram-negative LPS-induced sepsis) and was ultimately lethal, did nothing significant in TLR4-deficient mice, despite being in similar numbers in the tissues (spleen, lungs, kidney) of the two types of mice; there was also no evidence of the microbe invading deep into the tissue. The less virulent strain, similar in number in both mouse types, but overall lower (= better clearance), had no significant effect on these mice.
Therefore, regardless of virulence, A. baumannii didn’t actually invade the tissues to cause host damage. So what differentiated between highly virulent (=more damage) and less virulent (=relatively less or no damage) strains? The authors compared a panel of clinical isolates of the bacteria. It didn’t appear that LPS density on the microbe was related to strain virulence in vivo, but the authors discovered that the amount of LPS shed during growth of the strains was strongly correlated to the strain’s potency to induce TLR4 activation. In other words, the most virulent strains shed the most LPS during growth in vitro (and presumably in vivo, too) and correspondingly caused maximum TLR4 activation.
I hope you’ve understood the significance of that observation, gentle reader. It seems to indicate that if there is a means to stop the LPS from being shed or produced, the virulence of the A. baumannii strain may perhaps be attenuated. And that’s exactly what the authors found out: “Inhibition of LPS biosynthesis did not kill A. baumannii but enhanced opsonophagocytosis and decreased inflammation, resulting in protection of mice from lethal infection. Which means, that shutting down the production of LPS did not kill the bacteria in the traditional way (the way, say, an antibiotic works), but reduced the excessive inflammation in the host, giving a chance to host immune cells, such as neutrophils and macrophages, to destroy the bacteria.
So, how is LPS biosynthesis inhibited? A few years ago, a key enzyme – a zinc-dependent metalloamidase – in LPS biosynthesis was identified; the enzyme, produced by an essential, single copy gene, is referred to as ‘LpxC’, a.k.a. UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase – charming name, isn’t it? – and it catalyzes the committed step of synthesis of lipid A (the lipid component of LPS) in virtually all Gram-negative bacteria . Since then, given the role of LPS in the pathogenicity of many Gram-negative organisms, LpxC has been designated a good target for developing therapeutics for these group of microbes, and several small molecule inhibitors of this enzyme have been designed and studied. The study by Lin et al. used an investigational LpxC inhibitor that is in advanced preclinical development, called LpxC-1.
LpxC: Image modified for educational purpose from Figure 2 of
Barb AW and Zhou P, Curr Pharm Biotechnol, 2008, 9(1): 9-15.
LpxC inhibitors have been around for a short while and have been studied by many investigators. But the beauty of this study by Lin et al. lies in (a) connecting an essential bacterial component, LPS, to virulence in a novel way, i.e., via quantity released; and (b) recognizing the therapeutic importance of LpxC inhibitors in reducing host immunopathogenesis (‘over-reaction by the host body’) as well as bacterial virulence – even in absence of any bacteria-killing (‘bactericidal’) activity – by simply lowering LPS quantity. This study also opens the door to other related and important investigations; as the authors conclude:
The molecular genetics and structure of LPS that result in greater shedding by the more-virulent strains merits investigation, since elucidating these factors should result in novel targets for therapeutic intervention.
- Lin Lin, Brandon Tan, Paul Pantapalangkoor, Tiffany Ho, Beverlie Baquir, Andrew Tomaras, Justin I. Montgomery, Usa Reilly, Elsa G. Barbacci, Kristine Hujer, Robert A. Bonomo, Lucia Fernandez, Robert E. W. Hancock, Mark D. Adams, Samuel W. French, Virgil S. Buslon, & Brad Spellberg (2012). Inhibition of LpxC Protects Mice from Resistant Acinetobacter baumannii by Modulating Inflammation and Enhancing Phagocytosis mBio, 3 (5) DOI: 10.1128/mBio.00312-12
- Casadevall A, & Pirofski LA (2003). The damage-response framework of microbial pathogenesis. Nature reviews. Microbiology, 1 (1), 17-24 PMID: 15040176
- Cerqueira GM, & Peleg AY (2011). Insights into Acinetobacter baumannii pathogenicity. IUBMB life, 63 (12), 1055-60 PMID: 21989983
- Barb AW, & Zhou P (2008). Mechanism and inhibition of LpxC: an essential zinc-dependent deacetylase of bacterial lipid A synthesis. Current pharmaceutical biotechnology, 9 (1), 9-15 PMID: 18289052
In the first part, I introduced the Dendritic Cells (DCs) as immune sentries entrusted with a surveillance function, and mentioned how HIV is able to subvert the normal functions of DCs and and use them as Trojan Horses to infect CD4+ helper T-cells. I also referred to a 2009 study which discovered a possible involvement of certain membrane lipids in the process of DC-mediated HIV trans-infection of T-cells.
Dendritic Cells (DCs) are important members of the mammalian immune system. Working at the interface of innate and adaptive immune response, DCs are primarily antigen-presenting cells (APCs). DCs are derived from certain hematopoietic (bone-marrow derived) progenitors of either lymphoid or myeloid lineage, giving rise, respectively, to plasmacytoid DCs (pDCs) and myeloid DCs (mDCs) that localize to mucosal epithelium (inner lining of nose, lungs, the GI tract; also, the langerhans cells of the skin), as well as to peripheral blood.
I am tremendously psyched about this fascinating report published in Blood about a week back. The paper from this German multi-institutional group describes how, in an HIV-infected leukemic patient, transplantation with CCR5Δ32/Δ32 stem cells appeared to cure HIV. Even as I write this, I can barely contain my excitement; this finding has tremendous possibilities.
For the uninitiated, cellular chemokine receptors (CCR), as well as C-X-C Chemokine receptors (CXCR), are cell-surface protein molecules that ordinarily bind to chemokines (small protein molecules that act as chemo-attractants to guide the migration of cells for various purposes), activating signalling processes downstream into the cell. HIV, the human immunodeficiency virus, co-opts such receptors, most commonly CCR5 or CXCR4, to mediate its entry into its primary target, the CD4 T-lymphocytes. Viral replication in these cells utilizes the cellular gene expression processes, which are, understandably, most active in activated cells; therefore, HIV infection targets activated CD4 T-cells, especially the activated memory CD4+ T-cells in gastrointestinal mucosa, leading to their loss in the peripheral blood and lymphoid tissues, gradually destroying the immune system.
The aim of the currently available anti-retroviral therapies (ART, including ‘Highly Active ART’ or HAART) is to control the HIV replication, thereby allowing the immune system to be restored – which delays the disease progression. However, clever, little HIV establishes reservoirs of latently infected CD4+ T-cells and tissue phagocytes (such as macrophages and glial cells), as well as anatomical niches, such as the prostate gland, that continues to harbor the replication-competent virus despite ART – which allows for re-infection (technically, re-activation) once the ART is discontinued.
However, a multicenter cohort study, published in Science in 1996, made an important observation: Individuals whose cells contained two copies (homozygous) of a deletion variant (Δ32) of the CCR5 gene (CCR5Δ32/Δ32) – which abrogated cell surface expression of CCR5 – were naturally resistant to infection by CCR5-recognizing HIV strains. Moreover, progression to full blown AIDS was delayed in HIV-infected individuals by more than 16 years (the so-called ‘Long Term Progressors’) if they were homozygous for the Δ32 mutation, and by 2-4 years if they were heterozygous (i.e. had one copy of the mutated gene).
The German group conducting the current study had previously documented the absence of rebound viremia (“virus in blood”) in the first 20 months after cessation of ART in their HIV-infected patient who received CCR5Δ32/Δ32 stem cells as treatment for relapsed acute myeloid leukemia (AML). This report raised the possibility of using transplantation of selected or transgenic hematopoietic stem cells as a treatment for HIV/AIDS.
However, in an already HIV-infected patient, ordinarily the pre-transplant conditioning (immune suppression, i.e. destruction of the transplant recipient’s immune system in order to prevent rejection, as well as cytoreduction, i.e. the depletion of host cells – by means of intense chemotherapy and/or radiation) process doesn’t lead to the complete elimination of HIV from the tissue reservoirs despite HAART, and such patients have been known to experience rebound viremia from the pre-transplant HIV population.
Therefore, in the current study, the German group looked for several parameters in their patient, namely:
(a) Immune reconstituion, i.e. the restoration of CD4+ T-cells in the patient. They evaluated the reconstitution in the systemic circulation, as well as in the mucosal immune system, for >3.5 years after the transplant.
(b) Continued HIV-susceptibility of the recovered immune cells. They analyzed the activation status and CXCR4 expression profile of the recovered CD4+ T-cells, as well as their susceptibility to a productive HIV infection.
© Stability and persistence of the latent HIV-reservoir during the period of immune reconstitution following the CCR5-mutated stem cell transfer. They examined different tissue compartments for donor (CCR5-negative) and host (CCR5-positive) immune cells by immunofluorescence microscopy as well as CCR5-genotyping.
This brings us to the most amazing part of their findings. They observed that:
1. Host T-cells, including the long-lived HIV target cells of host-origin, were completely eliminated from the periphery following the transplant, being replaced by donor derived cells. This is an exceedingly important observation, since non-circulating immune cells (tissue T-cells and monocyte/macrophages) are virtually resistant to chemo/radio-therapy, and their elimination ensures removal of possible viral reservoirs. They found no CCR5 expressing host-origin cells in the liver, colon and brain within two years.
2. Numbers of donor-derived peripheral CD4 T cells increased continuously reaching healthy levels in two years, along with an enrichment of memory, as well as naïve, central, CD4 T cells. Results from their control groups established that the T-cell recovery was primarily through the homeostatic proliferation of memory CD4+ T cells.
3. Circulating donor-derived CCR5-negative CD4+ T cells were efficiently recruited to the gastrointestinal tract, repopulating the mucosal T-cell compartment.
4. Over 45 months following transplantation, HIV RNA and DNA remained undetectable in tissue compartments, and HIV-specific antibodies in the serum gradually decreased over time.
5. Importantly, however, the recovered, donor-derived CCR5-negative CD4+ T cells maintained the level of CXCR4 expression, thereby remaining vulnerable to HIV-variants that target this molecule (called ‘X4 HIV’), which they also demonstrated experimentally.
Therefore, the CCR5Δ32/Δ32 stem cell transplantation in this HIV-infected AML patient was instrumental in successful recovery of CD4+ T cells and complete elimination of HIV, as well as HIV reservoirs, from the patient. This was likely possible because of their three-pronged approach: (a) The HAART was active against the virus, (b) the pre-transplant conditioning effectively reduced the peripheral HIV-infected cells, and © the stem-cell transplant effectively flushed out the remaining HIV-reservoirs, while remaining impervious to fresh infection.
As with any scientific study, this report, too, comes with caveats that temper the enthusiasm.A The most critical of such caveats is that these were observations in just a single patient. Secondly, the CCR5-negative CD4 T cells, and thereby the patient, still remained susceptible to X4 HIV. This patient was possibly extremely fortunate to have been infected with the CCR5-tropic variant (‘R5’) of HIV, and not X4, and also not with a variant that mutated rapidly to lose its CCR5-tropicity. In addition, although by common prognostic markers (i.e. plasma viral load and peripheral CD4 T cell count) shows absence of HIV-disease in this patient, presence of still-latent HIV in distinct tissue compartments, especially the hard-to-reach mucosal immune system, cannot still be completely discounted. Also important to remember is the fact that the bone-marrow transplant (hematopoietic stem cell transplant) process is inherently risk-laden, and still associated with significant mortality and morbidity.
But all in all, the fact that the stem-cell transplantation intended to treat AML has managed to keep this patient HIV-free for 3.5 years in absence of continued ART represents a powerful and exciting achievement in the continuous war against the scourge of HIV, opening up distinct possibilities for future HIV-therapy. One such possibility might be the voluntary, pro-active banking of autologous (from the same individual) stem cells, perhaps even with an engineered CCR5 mutation, that can be used later, in the event of an HIV-infection, or certain cancers. It appears that such banking services are already in operation, albeit currently of doubtful utility.
1. Allers K, et al. Evidence for the cure of HIV infection by CCR5Δ32/Δ32 stem cell transplantation. Blood. 2010 Dec 8. [Epub ahead of print] PMID: 21148083 This is paper under discussion.
2. Chun TW, Fauci AS. Latent reservoirs of HIV: obstacles to the eradication of virus. Proc Natl Acad Sci U S A. 1999 Sep 28;96(20):10958-61. PMID: 10500107.
3. Smith DM, et al. The prostate as a reservoir for HIV-1. AIDS. 2004 Jul 23;18(11):1600-2. PMID: 15238781.
4. Dean M, et al. Genetic restriction of HIV-1 infection and progression to AIDS by a deletion allele of the CKR5 structural gene. Hemophilia Growth and Development Study, Multicenter AIDS Cohort Study, Multicenter Hemophilia Cohort Study, San Francisco City Cohort, ALIVE Study. Science. 1996 Sep 27;273(5283):1856-62. PMID: 8791590.
5. Hutter G, et al. Long-term control of HIV by CCR5 Δ32/Δ32 stem-cell transplantation. N Engl J Med. 2009;360(7):692-698. PMID: 19213682.
Note: A. A fact most purveyors of pseudoscience like to capitalize on, without understanding the dynamic, empiricism-driven, evidence-based nature of science.
I have a blog. On Nature Network. Fancy that!
I must clarify, though, that the indicated ‘suddenness’ is procedural, rather than temporal. Richard Grant (yes, that* Richard; Hat-tip to you!) had planted the seed of an idea – of getting my own blog where I can rant and rant and… (did I say, ‘rant?’) to my blessed heart’s content. Taking advantage of the confusion and hassles over moving the NN blogging platform to MT4, I sneaked in. I mean, that must be it. Otherwise – think about it – why would they grant me access to a hallowed area already populated by heavyweights? (Ahem! Metaphorically speaking, of course!)