Friday, November 20, 2015

Global Antibiotic Resistance Alert: MCR-1 in Plasmid


Antibiotic resistance is a topic of great interest and perhaps one of the most talked about topic in this blog. One of the most talked about resistance in last couple of years was the resistance to carbapenem class of drugs. It then became a massive issue with emergence of strains called as NDM (New Delhi Metallo Beta Lactamase). The use of this name is still debated. Important point is we now live in an era of superbugs, and rapidly running out of options.

Gram negative bacteria, especially Enterobacteriaceae members are one of the most difficult to treat pathogens, since they are known to acquire resistance quickly especially through plasmid mediated transfer. When resistant to everything else colistin is considered as the last option. Colisitn (Polymixin E) was discovered in 1947 from Paenibacillus polymyxa. Chemically, Colistin is a mixture of cyclic polypeptides colistin A and B. However, it was rarely used due to its toxic properties. When medicine ran out of options, the drug was repurposed. But colistin resistance was not uncommon. Several bacteria have innate resistance to colistin. Some bacteria have recently developed resistance colistin resistance through target modification or an enzyme- Colistinase. Klebsiella pneumoniae strains resistant to colistin is a story of the past.

Fig 1: Structure of Colistin.
The exact mechanism of colistin activity has not been described. It is proposed that Colistin being cationic in nature binds well to the polyanionic bacterial LPS and acts through a detergent-like effect, via a two-step mechanism. Colistin activity is not dependent on the metabolic activity of the bacteria, thus making it a great antibiotic. It is argued this property also makes it difficult for the bacteria to acquire resistance. Interestingly, the genes coding for resistance was described only in the chromosome and hence resistance transfer was considered minimal possibility. But colistin resistance has been increasingly reported though the prevalence varies from region to region.

The latest reported published in Lancet infectious disease has once again brought this topic into headlines. In a routine resistance survey, the team noted that MCR-1 was present in an E coli (strain SHP45) plasmid. The paper reports that the MCR-1 gene was present in E coli isolates collected from 78 (15%) of 523 samples of raw meat and 166 (21%) of 804 animals during 2011–14, and 16 (1%) of 1322 samples from inpatients with infection. Further mechanistic studies showed that this gene codes for phosphoethanolamine transferase enzyme, leading to addition of phosphoethanolamine to lipid A, thus changing colistin target. The studies indicate that this is a jump from animal, since use of colistin is so common in animal farming in China

Co-author Jian-Hua Liu expressed the findings as worrying "The last class of antibiotics in which resistance was incapable of spreading from cell to cell." Another researcher Timothy Walsh comments, "If MRC-1 becomes global, which is a case of when not if, and the gene aligns itself with other antibiotic resistance genes, which is inevitable, then we will have very likely reached the start of the post-antibiotic era. We must all reiterate these appeals and take them to the highest levels of government or face increasing numbers of patients for whom we will need to say, 'Sorry, there is nothing I can do to cure your infection." A yet another co author Jianzhong Shen says, "Because of the relatively low proportion of positive samples taken from humans compared with animals, it is likely that mcr-1 mediated colistin resistance originated in animals and subsequently spread to humans"

Currently, a lot of scientists are recommending global surveillance and stop polymixin use in animals. Certain scientists also raised hope by pointing to earlier emergence of Plasmid mediated Vancomycin resistant S aureus which didn't globalize. Patel from CDC comments, “That’s our classic example where we were very, very concerned and then it didn’t spread”. But its well acknoweledged by everyone that desperate multi level control of antibiotic and surveillance will help in limiting the spread.
Bialvaei AZ, & Samadi Kafil H (2015). Colistin, mechanisms and prevalence of resistance. Current medical research and opinion, 31 (4), 707-21 PMID: 25697677

Liu, Y., Wang, Y., Walsh, T., Yi, L., Zhang, R., Spencer, J., Doi, Y., Tian, G., Dong, B., Huang, X., Yu, L., Gu, D., Ren, H., Chen, X., Lv, L., He, D., Zhou, H., Liang, Z., Liu, J., & Shen, J. (2015). Emergence of plasmid-mediated colistin resistance mechanism MCR-1 in animals and human beings in China: a microbiological and molecular biological study The Lancet Infectious Diseases DOI: 10.1016/S1473-3099(15)00424-7

Tuesday, November 17, 2015

Dead bacteria fights Lab model of Colorectal cancer


One of the greatest focus on research in medicine is currently cancer treatment strategies with companies interested to shell out millions over research. However, it has been a very difficult to beat disease. Just like in the case of infections, cancer cells often turn out to become resistant to treatment drugs thanks to its ability to mutate too fast. Also, it has become clear in recent years that several infections such as oncogenic virus and some bacteria have a role in oncogenesis or development of cancer.

Table 1: Oncolytic viruses in development.
Of late, one of the treatment strategies that has been tried and tested is a therapeutic called as Oncolytic virus therapy. Though this technology has a scope for improvement, recently one of them - Talimogene laherparepvec (T-VEC) which is a version of the herpes simplex virus (Officially known as IMLYGIC™) is approved by FDA for Clinical use in metastatic melanoma, raising hope for this strategy. There are several such viruses in current research (Shown in Table 1).

One of the top concerns in cancer research is colorectal carcinoma. With 1.4 million new annual cases worldwide, it stands one among the top in the world. The options for treatment is also quite low. Also in this case chemotherapy and radiotherapy don't work well due to hypoxic condition often seen in this particular tumour environment.

Scientists from Nanyang Technological University argued that the environment is right for growth conditions of anaerobic bacteria. Based on previous reports, they further argued that clostridium sporogenes which is known to accumulate near tumor cells is an excellent candidate. Being a proteolytic species, it can also cause cell death. But there is an issue. Once the infection is established, though tumor cells are rapidly killed it can cause frank infection which can be devastating. Recently, a study was conducted using C novyi strain against rat brain tumours and a variety of solid tumors in dogs. The success lead to a further human clinical trial on a patient with late-stage metastatic retroperitoneal leiomyosarcoma. Though tumor was downsized effectively, infective complications appeared which was managed with antibiotics.

Based on the above, heat killed C sporogenes was tested in a tumor model (3D tumor model), and found that within 72 hours, nearly 74% of the virus was destroyed. The team also tested bacterial secretions on tumor cells showing a significant effect. In a sense, the dead bacteria continues to secrete degrading compounds which is killing the cells. I see some problems that needs to be addressed here. It is not clear to me what the bacterial secretion include. So, it would be interesting to know what happens in context with normal cells. In case of alive bacteria, the tumor specificity of bacteria ensures sparing normal cells to an extent.

One of the author comments "“We found that even when the C sporogenes bacteria is dead, its natural toxicity continues to kill cancer cells, unlike the conventional chemotherapy drugs which need oxygen to work. While other research groups have experimented with bacteria therapy to destroy cancer cells, the biggest problem is that live bacteria will grow and proliferate, posing a high risk of infection and increased toxicity to patients. In our study, as the bacteria were already killed by heat, there was no risk of the bacteria multiplying and causing more harm than the desired dose meant to kill colorectal cancer cells". James Best, Dean of NTU says, “This is a significant discovery that potentially opens a new avenue to tackle this very common cancer, which is difficult to treat after it has spread. While it is early days, this exciting research finding provides hope of a new treatment option for millions of people affected by bowel cancer each year.” Source
Dolgin E (2015). Oncolytic viruses get a boost with first FDA-approval recommendation. Nature reviews. Drug discovery, 14 (6), 369-71 PMID: 26027526

Roberts NJ, Zhang L, Janku F, Collins A, Bai RY, Staedtke V, Rusk AW, Tung D, Miller M, Roix J, Khanna KV, Murthy R, Benjamin RS, Helgason T, Szvalb AD, Bird JE, Roy-Chowdhuri S, Zhang HH, Qiao Y, Karim B, McDaniel J, Elpiner A, Sahora A, Lachowicz J, Phillips B, Turner A, Klein MK, Post G, Diaz LA Jr, Riggins GJ, Papadopoulos N, Kinzler KW, Vogelstein B, Bettegowda C, Huso DL, Varterasian M, Saha S, & Zhou S (2014). Intratumoral injection of Clostridium novyi-NT spores induces antitumor responses. Science translational medicine, 6 (249) PMID: 25122639

Lokody, I. (2014). Anticancer therapy: Bacterial treatment for cancer Nature Reviews Drug Discovery, 13 (10), 726-726 DOI: 10.1038/nrd4447

Bhave MS, Hassanbhai AM, Anand P, Luo KQ, & Teoh SH (2015). Effect of Heat-Inactivated Clostridium sporogenes and Its Conditioned Media on 3-Dimensional Colorectal Cancer Cell Models. Scientific reports, 5 PMID: 26507312

Thursday, November 12, 2015

Toxoplasma Vaccine: A step Closer


Toxoplasma gondii is a topic about which I have posted several times in this blog space. The ability of this protozoa to infect a wide range of species including humans is astounding. Felines are expected to be the most common host, and classically acquired by ingestion of contaminated source it can also spread through several other means. T. gondii can only sexually reproduce in the gut of felids. Its ability to be latent without any apparent manifestation has been considered important. On a global scale, toxoplasmosis is expected to be present in roughly 22% of healthy population. Multiple studies has shown that a proportion of healthy blood donors are serologicaly positive. This speaks for itself.

Fig 1: Toxoplasma invasion into the cell.
The most common route of infection is through ingestion, where the invasion begins by entering into enterocytes. It can also breach through epithelial layers. Ultimately, the ability to be motile and circulation through lymphatic system allows the pathogen to invade any kind of tissue.

Toxoplasma stays inside a self created vacuole called the parasitophorous vacuole (PV) which prevents endolysosomal fusion. The process of cellular entry is complex in terms of molecular interactions. The rhoptry of toxoplasma secretes several proteins to aid the process. Some proteins also travel to the nucleus to modulate host cell activity. PV serves as a safe house for the parasite. Surprising it may sound, PV recruits host mitochondria and endoplasmic reticulum providing the required nutrient support for the parasite.

Toxoplasma immunology is a little messy and not completely understood (Oh!! You could say nothing is completely understood in biology). The best defense for the cell is to disrupt the PV thus exposing the toxoplasma to immunological attack. This attack is best tailored through IFN-γ, which can activate cellular immune cascades. The best defined molecules in action include IFN-γ induced GTPases- immunity-related GTPases (IRGs) and guany-late-binding proteins (GBPs). Toxoplasma possess counter acting molecules. Rhoptry molecules especially ROP18 and ROP5 are of great interest. ROP5 is a pseudokinase which modulates the catalytic activity of ROP18 which in turn effect the phosphorylation of immune proteins such as IRGs (such as Irga6, Irgb6, and Irgb10). This effectively silences immune responses. Basically, ROP5/ROP18 increases the survival time of the parasite inside IFN-γ activated cells especially macrophages.

Fig 2: Role of p62 in immune
response against Toxoplasma.
Needless to say, there is a need for good and high IFN-γ. New research has spread some light. The team led by Masahiro Yamamoto showed that p62, a host molecule, has a potential role in exerting immune effects of an experimental pathogenic parasite toxoplasma-inactivated vaccine. P62 also known as Sqstm1 (Sequestosome 1) is one of the well known molecule acting as a selective autophagy adaptor involved in autophagy and intra-cellular immunity. The study in summary shows that P62 and ubiquitination of PV is important for getting the immunity right.

Despite the ubiquitous nature of Toxoplasmosis, there has been no effective vaccine developed till date. There are several reason's for why we don't have a candidate vaccine. some of them include, lack of basic knowledge on candidate targets and immunology. This study pushes this void a little bit further and perhaps also provides us an idea on potential targets to be looked at. Oh yes, We are a step closer.
Hunter CA, & Sibley LD (2012). Modulation of innate immunity by Toxoplasma gondii virulence effectors. Nature reviews. Microbiology, 10 (11), 766-78 PMID: 23070557

Behnke MS, Fentress SJ, Mashayekhi M, Li LX, Taylor GA, & Sibley LD (2012). The polymorphic pseudokinase ROP5 controls virulence in Toxoplasma gondii by regulating the active kinase ROP18. PLoS pathogens, 8 (11) PMID: 23144612

Youngae Lee, Miwa Sasai, Ji Su Ma, Tatsuya Saitoh, Shizuo Akira, Masahiro Yamamoto (2015). p62 Plays a Specific Role in Interferon-g-Induced Presentation of a Toxoplasma Vacuolar Antigen
Graphical Cell Reports, 13 (2), 223-233

Liu Q, Singla LD, & Zhou H (2012). Vaccines against Toxoplasma gondii: status, challenges and future directions. Human vaccines & immunotherapeutics, 8 (9), 1305-8 PMID: 22906945

Monday, November 02, 2015

Lab series# 8: PBMC Isolation technique


Immunological studies often requires a set of cells called as Peripheral blood mononuclear cells or what is commonly known as PBMC. PBMC research currently forms one of the platforms for immunological research. Molecular pathways of inflammation, transcriptional activity etc in response to infections, antigens and many more form the basis of understanding modern molecular immunology. I was recently discussing regarding PBMC isolation with a friend and he told me that its not a very common knowledge as I had expected. So let us take a quick review.

Table 1: T cell population in PBMC
Whole blood obtained from venipuncture has many different types of cells in it. The RBC, WBC and platelets. RBC forms a huge major bulk. PBMCs primarily include lymphocytes (60-90%), monocytes (10-30%), and dendritic cells (<2%). Within the lymphocyte population the sub populations exists in variable concentration as shown in Table 1. Normally, PBMC's are devoid of granulocytes. For experiments requiring the use of Neutrophils or Eosinophils there is a different way of doing it.

The most popular technique used for PBMC isolation is the Ficoll-Paque density centrifugation method. Principle of the method is as follows. Anti-coagulated blood, in Ficoll leads to differential migration of cells during centrifugation resulting in the formation of multiple layers as shown in Fig 1. The lymphocyte and monocyte population with denisty of 1.077g/ml forms a layer, cells in which is called as PBMC. Basophils density can vary and are hence sometimes present in this layer.

Fig 1: Layers obtained after Ficoll
gradient centrifugation.
The most important component of this whole procedure is the Ficoll-paque (Histopaque). The contents of this solution include- Ficoll, Diatrizoate Sodium, Edetate Calcium Disodium. Ficoll is a neutral, highly branched, high-mass, hydrophilic polysaccharide which dissolves readily in aqueous solutions. Ficoll PM400 and PM70 are high molecular weight sucrose-polymers formed by copolymerization of sucrose with epichlorohydrin. P400 is considered better than P70. P400 because of its high molecular weight (~ 400 000) and low content of dialyzable material does not normally penetrate biological membranes. Also by itself, PM400 can act as a cryoprotectant. For a detailed contrast between PM400 and PM70, check this article by GE Life science.

Before using the whole blood, for procedure in my experience, yield is better when you reduce other components. First centrifuge the whole blood and remove as much plasma as possible. With little expertise I have seen some people also are able to remove a lot of sedimented RBC. The rest of the blood sample is diluted 1:1. For dilution usually balanced salt solution or PBS is recommended. In a separate tube add about 15 ml of Ficoll solution and allow it to come to room temperature. Slowly add the diluted blood sample through sides of the tube such that blood overlays the Ficoll solution. Believe me, this step is crucial. The slower and more gentle is the addition, better is the results. Centrifuge the tube at 800g for 25 min at room temperature and remove the cells from layer into a fresh tube. Wash the cells with balanced salt solution or PBS. Done properly recovery rate is more than 95% with good viability.

The one disadvantage of this method is the need to be very careful during the overlaying of diluted blood on Ficoll. This aspect has been improved upon by techniques such as Sepmate which creates a barrier between Ficoll and blood thereby allowing rapid pouring. A method that reached the farthest, is a vacutainer tube called as CPT (Cell preparation tube). This is a one step process, where blood is collected in tube, centrifuge and take the cells out. Studies indicate that there is no significant difference in quality of cells extracted by any of the methods, though they reduce the time required.

For studies where Neutrophil is required, the bottom layer is extracted containing RBC and Neutrophils. RBCs are then specifically lysed using methods such as Dextran- NaCl method.

Rarely it happens that the PBMC are processed for whatever is the experiment immediately, though that would be the ideal case. PBMC are best preserved in RPMI 1640 or FBS with DMSO. The best freezing condition of PBMCs is 40% FBS with 10-15% DMSO (i.e. 12.5%) with cooling rate of 1ºC/ minute is recommended. For long-term storage freezing in liquid nitrogen is best. Even with excellent storage the viability after thawing would be only about 70-80%.
Mallone R, Mannering SI, Brooks-Worrell BM, Durinovic-Belló I, Cilio CM, Wong FS, Schloot NC, & T-Cell Workshop Committee, Immunology of Diabetes Society (2011). Isolation and preservation of peripheral blood mononuclear cells for analysis of islet antigen-reactive T cell responses: position statement of the T-Cell Workshop Committee of the Immunology of Diabetes Society. Clinical and experimental immunology, 163 (1), 33-49 PMID: 20939860

De AK, Roach SE, De M, Minielly RC, Laudanski K, Miller-Graziano CL, & Bankey PE (2005). Development of a simple method for rapid isolation of polymorphonuclear leukocytes from human blood. Journal of immunoassay & immunochemistry, 26 (1), 35-42 PMID: 15754803

Oh, H., Siano, B., & Diamond, S. (2008). Neutrophil Isolation Protocol Journal of Visualized Experiments (17) DOI: 10.3791/745

Nazarpour R, Zabihi E, Alijanpour E, Abedian Z, Mehdizadeh H, & Rahimi F (2012). Optimization of Human Peripheral Blood Mononuclear Cells (PBMCs) Cryopreservation. International journal of molecular and cellular medicine, 1 (2), 88-93 PMID: 24551763