Abstract
Macrophages, the executioner of phagosome maturation, are central to coordinate and cooperate as the bridge between innate and acquired immunity. Mice primed with attenuated Leishmania promastigote showed host defense, such as total protection against LPS-induced endotoxic shock and, diarrhoeagenic E. coli lethal infection. Cell-based empirical preparations and isolated lipids, sphingolipids and lipoproteins were made out of the promastigotes. Host macrophage-mediated enhancement of microbicidal actions, non-specific and specific host immunity boosting and mitigation of antomicrobial resistance by the empirical preparations and, the cancer cell apoptosis, resolution of sepsis, combating autoimmune disease by isolated chemical constituents were evident, respectively. Macrophage phagosome maturation is the key factor of all these changes and indeed the attenuated Leishmania promastigote was found as an efficient agent for such maturation. To assess clinical impact of the studies, the therapeutic aspects of isolated total promastigote lipid were investigated on the synovial fluid mononuclear cells of RA (rheumatoid arthritis) patient as a case study including an animal model of the disease in parallel. The use of the attenuated Leishmania promastigote to produce human therapeutic vaccines that served Indian people for decades (1954–2005) by a nearly unknown Kolkata (India) based firm (IBL) was rediscovered recently.
Keywords
- leishmania promastigote
- macrophage
- therapeutics
1. Introduction
‘There is at bottom only one genuinely scientific treatment for all diseases, and that is to stimulate the phagocytes’. Gorge Bernard Shaw in ‘
An attenuated
1.1 The attenuated leishmania promastigote and watery diarrhoea control
The bacteria
Intestinal macrophages inherently produce both pro-and ant-inflammatory cytokines including the anti-inflammatory IL-10. Macrophages are M1 phenotype when coming in contact with attenuated
1.2 Role of empirical preparations of the attenuated Leishmania promastigote on non-specific host defence
The preparations were made of 106–107 promastigote per ml. cell count based on six different lots and then used for macrophage stimulation in an
The parasite
Cell homogenate or CH appeared most potent than the IC (intact cell). The macrophage response modulation by the IC, CH and CD resulted in generation of both the reactive oxygen species, ROS and nitrogen intermediate NO significantly but not as high as that of LPS or TNF-α associated induction. The LPS stimulation was very sharp and massive compared to IC and CH effect. However, these were not as sharp as the action of LPS in terms of TNF-α release and time kinetics. When the LPS response was suppressed in combination with IC or CH preparations, the ultimate product formation to TNF-α was blocked somewhere at an intermediate step before reaching the penultimate step. But the effect of externally added TNF-α along with IC or CH, in terms of total cytokine release, indicated an additive effect. This observation was of critically significant because once the LPS response attained its ultimate manifestation to TNF-α, its formation has not interfered further with respect to the presence of promastigote components. Macrophages pre-treated with IC, CH and CD did not respond to NO generation by LPS stimulation. The fraction CD or cell debris possibly contained DNA, RNA, insoluble membrane glycoprotein, glycolipids, lipids and lipoprotein, primarily a combined mixture of all these. DNA has no stimulatory or response modifying effect on LPS activity so the blocking of LPS action on macrophage activity by lipids and/or lipoprotein might be proposed and it was indeed the case as was evident subsequently.
IC, CH and CD pre-exposed macrophages showed higher phagocytic activity and intracellular killing of bacteria. This was confirmed by demonstrating in the IC mediated enhanced phagocytosis using engulfed GFP
The
2. Phagosome maturation by the attenuated Leishmania donovani (UR-6 MHOM/ 1978) promastigote
Phagosome maturation is essentially the most crucial biological process that performs the engulfment of the microbial pathogens and then degrades it to clear the infection. Macrophages are the key effector cells to furnish all the jobs successfully through the orchestration of its phagosome functions. Thus, the macrophages carry out two functions, one is phagocytosis as the ‘housekeeping’ or scavenging function and the other one is meant for host defence. Particularly the same macrophage has to perform both the functions to achieve a successful host defence strategy. There is also another aspect of phagosome maturation, the maintenance of tissue homeostasis, a determinant to recon as the basic function of macrophage assigned to the fulfilment of phagosome maturation. The removal of apoptotic cells and other extracellular component derived as by-products are a daunting task that is to be completed by macrophages. These are also the responsibilities of mature phagosomes and occur during the process of maturation. In case of the apoptotic cells, they display well known ‘eat me’ signals which recognizes various type of collectins, scavenger receptors, integrins and bridging molecules that link the surface structures to the macrophage receptors. The viable cells express specialized receptor such as CD47 that acts to inhibit the phagocytosis through receptors such as SIRP-α as apoptotic cell undergo silent absorption instead of degradation and elimination.
Thus, the phagocytosis of both microbes and apoptotic cell is carried by the same macrophage but there is a fundamental difference. The apoptotic cell phagocytosis by macrophage is trigged by anti-inflammatory responses, such as through the production of growth factor (TGF)-β while, in contrast the microbial cell phagocytosis occurs upon triggering the inflammatory response through production of TNF-α, IL-1 and IL-6 and make alert to the infection. The objective of the phagosome maturation by intact promastigote of attenuated
2.1 Phagocytic phase of the phagosome maturation by attenuated Leishmania promastigote
This is the demonstration of the phagocytosis associated events that were described with the empirical preparations of the attenuated
The macrophage exposed to the heat-killed intact attenuated
The heightened level of the expression and translocation of NF-kB and c-Jun, the crucial transcription factors were evident due to the attenuated
The increase in pro-inflammatory cytokine activity was mediated through p38MAPK and p44/42 MAPK activation. The enrichment of the attenuated promastigote exposed macrophage-mediated release of Th1 type cytokine over the Th2 type was observed as indicated by higher level of IL-12 over IL-10 though both the MAPK were involved here simultaneously. Other markers of the phagosome maturation are indicated by the co-localization of Rab7, Lysosomal Associated Membrane Protein-1, Cathepsin D, Rab 9 and V-ATPase. Inhibition of V-ATPase was found to cause significant hindrance in phagosome maturation. Acidification and phagosome maturation, a coupled phenomenon occurs in unison in the attenuated promastigote exposed macrophages ([8] and ref. therein). Thus, the achievements of macrophage phagosome maturation, the crucial functional attainment have been well documented with crucial manoeuvring capability using the promastigotes of attenuated
3. Impact of phagosome maturation as a macrophage game changer
Phagosome maturation is considered the end of the phagocytic process. In this process macrophages, the classical phagocytes, among others if properly activated, the efficient clearance is ensured. As it has already been evidenced that attenuated
The macrophages, as the pivotal host cells whose response modification through
4. Leishmanial lipid, macrophages and rheumatoid arthritis, an autoimmune disease
A cohort of patients in the department of Rheumatology, Calcutta Medical College, Kolkata, India, have fulfilled the criteria of RA (Rheumatoid Arthritis) as per the American College of Rheumatology [10] were selected for study. After approval of the medical college ethical committee and having patients’ consent Synovial Fluid (SF) collection was made from the patients showing knee joint swelling with signs of active synovitis. The adherent Synovial Fluid Mononuclear Cells (SFMCs) were then prepared and cultured in presence of total lipids isolated from the attenuated
The pathophysiological and therapeutic eventuality involving inflammation and cartilage destruction are of crucial significance of activated macrophages associated with the synovial membrane and knee joint. Permanent joint damage prompted a critical re-evaluation of therapeutic regimens currently used with anti-inflammatory and disease-modifying treatments for RA.
One of the major targets is the regulation of proinflammatory cytokines released by the monocytes –macrophage in Rheumatoid Arthritis or RA. TNF-α, here acts as pleiotropic cytokine reported to have an inductive effect on the enhanced expression of other cytokines, adhesion molecules etc. but mostly produced by macrophages in the synovial membrane. It is a proximal cytokine in the inflammatory cascade and the degree of expression depends on the histological configuration.
The leishmanial lipids were used to modulate responses of the macrophage of RA patients with a view to the resolution of the disease. In presence of the leishmanial total lipids, the aggravated inflammatory condition of SFMC was down-regulated showing its responses modification distinctly as if it was acting like a response modifying therapeutic agent. The response modifying, in terms of TNF-α release primarily was assayed experimentally both in an
The pro-inflammatory cytokines, namely TNF-α, IL-1β and reactive nitrogen intermediate NO (nitric oxide) and enzymes are abundantly released by the synovial tissue lining cell, synovial fluid cell and infiltrating monocyte–macrophage involved in driving the inflammatory response and joint destruction mainly [12]. So the study included estimation of the release of TNF-α, IL-1β and NO production by adherent SFMC, primarily the infiltrating monocytes-macrophage after treatment with the leishmanial lipids. An anti-inflammatory cytokines IL-10 was also induced by macrophage as it was evidenced during phagosome maturation and found to be released by the macrophage. So it was also included to be monitored whether countering the effect of the pro-inflammatory cytokines also takes place or not or could be quantifiable. During phagosome maturation, such anti-inflammatory cytokine, IL-10 release was observed by the intact attenuated
The adherent SFMCs, primarily monocyte-macrophage were stimulated with human gamma interferon (IFN-γ) or phorbol myristate acetate (PMA) before exposure to leishmanial lipids. The decreased release of the cytokine, TNF-α in the culture media confirmed the effect of leishmanial lipids as a suppressor. The distinctly diminished release of IL-1β and decreased NO production were found in parallel. The changes were observed with the lipids in a dose- and time-dependent manner. But an increased IL-10 release was established clearly as the effect of the promastigote lipids, demonstrating the leishmanial lipids functioned as an anti-inflammatory cytokine-releasing agents in a dose-dependent manner. Both the anti- and pro-inflammatory cytokine release were affected, though the release of the former increased while decreased secretion of the later favoured the relief or resolution of the disease. Thus, at the cytokine level, the lipid action was highly therapeutic in nature. With respect to the status of the transcription factor NF-kBp65 of SFMCs, a decrease in its level was evident in a dose-dependent manner. Thus, it was clear that the transcription factor expression in SFMCs of RA was also highly specific or sensitive to the presence of leishmanial lipids. The cytosolic protein content level was also suppressed by the leishmanial lipids in a dose-dependent manner.
The total viable cells present in adherent SFMCs of the RA patient as determined by MTT assay showed dramatic reduction of the viability in a dose- and time-dependent manner upon exposure to the leishmanial lipids. Sphingolipids comprise 5–10% of the leishmanial membrane lipids and it was shown that a sphingolipid-enriched preparation obtained from this attenuated
It has been reported that a higher expression of tm-TNF-α (transmembrane TNF-α) in RA patient resulted in an increase in apoptosis by tm-TNF-α, compared with healthy donors. After infliximab treatment, the tm-TNF-α binding was proposed as a regulator of the reverse signalling to improve the pathological condition and also as a result of cell-targeted therapeutic action [15]. Thus, the effect of leishmanial lipids was comparable to a cell-targeted therapeutic action in case of SFMCs of RA but no confirmed interaction could be proposed with tm-TNF-α binding. The advantage of higher dose (a dose 100μgm/ml) of leishmanial lipid could be exploited when higher (76–78%) apoptotic cell death of SFMCs of RA patients was observed indicating better recovery. SFMCs were diseased cells and not per say cancerous cells, and they acted as the target of leishmanial lipids.
Taking RA as representative case, the
5. Macrophage, lipids of attenuated Leishmania donovani promastigote, sepsis attenuation
The macrophage-mediated therapeutic applications based on the
5.1 The case of sepsis
Prospective experimental therapy vs.
An experimental therapy of sepsis has taken at first for discussion:
Mice not treated with pLTL (pathogenic Leishmanial Total Lipid) but challenged with LPS lethal shock had a survival rate of 41% and 9% at 24 hr. and 48 hrs, respectively, but displayed total death within 72 hrs. Mice primed for 3 consecutive days with pLTL at doses, 50 and 25 mg/ml and exposed to lethal dose of LPS had 78.8% and 53.8% survival without further loss of life. The reduced cytokine release was evident due to pTLT priming and factors [(IL-12p40, IL-17, IFN-γ, MIP-2, KC and RANTES (C–K)] were further included to prove its existence and they were detected by ELISA based assay. High dose pLTL, 50 mg/ml pre-treated animals showed the reduction of the vascular permeability factors, such as VEG, and suppressed the expression of cell adhesion molecules, including ICAM-1, VCAM-1, PECAM-1, P-selectin and E-selectin, compared with its level in liver of septic mice. Thus, endotoxin associated liver damage was improved considerably in the pLTL treated group.
Macrophages, the key regulators of the host immune response expression, play an important role in the pathogenesis of inflammation. They secrete quite a large number of inflammatory mediators such as prostaglandins, reactive oxygen and nitrogen species, inflammatory cytokines including tumour necrosis factor-alpha (TNF-α), interleukin-1 (IL-1), interleukin-6 (IL-6), interleukin-12 (IL-12), and interleukin-17 (IL-17), chemokines, including macrophage inflammatory protein (MIP), and bioactive lipids. These are regulated by the ubiquitous transcription factor, nuclear factor B (NF-kB). The consequences of complex immune reactions are described as sepsis that represents an uncontrolled inflammatory outburst from harmful host responses to infection, causing disruption and damage to several cells and tissues. IkB appears to function as a strong negative feedback mechanism elicitor that allows a fast turn-off of the NF-kB response to control inflammation-associated diseases. But identification of new therapeutic targets for the management of septic shock remains imperative as all investigational therapies, including anti-tumour necrosis factor (TNF-α) and anti-interleukin (IL-1) agents, have uniformly failed to lower the mortality of critically ill patients with severe sepsis. Though different bacteria have been identified as causative organisms in sepsis, gram-negative bacteria like
To decipher the molecular approaches by which LTL (Leishmania Total Lipids of attenuated promastigote) inhibits the inflammatory responses of Gram-negative bacterial sepsis, attempts were made to evaluate the survival rate and body weight improvement of mice in the
The recruitment of leukocytes at the inflammatory site required coordinated expression of specific combination of adhesion molecules and those are diverse in nature, sequentially develop to organize the pathophysiological condition with epithelial cells. The main endothelial CAMs(cell adhesion molecules cascade) involved in the inflammatory response are E-selectin and two members of the Ig-gene superfamily, intercellular adhesion molecule (ICAM)-1 and vascular cell adhesion molecule (VCAM)-1 that are expressed sequentially, but LTL addition alters the sequences of events towards attenuation. The study referred to is possibly the first evidence that attenuated leishmanial total lipid contributes to host defence during bacteremic sepsis caused by
5.2. The case of an experience-based therapy or a therapeutic vaccine for sepsis
It is said that if there is sepsis, an infection must be sought at a first instance. Then why there would be no vaccine? The term therapeutic vaccine means to get a patient vaccinated while in bed. The question is how it could be possible? The first dose would begin with 0.1 ml of the vaccine. This was a verified approach and practised over the decades.
The doctor who developed it was an M.D. degree awardee trained abroad (possibly in Germany) and provided it for treatment way back in early 40s of the last century and set up the first biomedical research institute in India. His name is
The author became interested in the vaccine because it works on the basis of macrophage functional modulation. This vaccine served the people here (India) for over 50 years (1954–2005) as per the document available. Name of the producer firm is
The documents were reached to the author in 2007, after 2 years of his retirement and
What were in the vaccines? The above bacteria, especially pathogenic but non-pathogenic types were also picked up and heat-killed preparations were made up. Then respective count was made by O.D. measuring against standard count in a spectrophotometer or any convenient equipment. Initial cell count was stated 100 to 10 million of the different bacteria and then suspended in homogenized leishmania promastigote (it was made of the promastigote of attenuated
This is a crude type of vaccine (based on whole cell) prepared long back and used till 2005 as per the documents available, and no detailed clinical data were recorded systematically. This is a separate chapter with the potential to revive the products though it was very primitive, but as per hearsay, it was very efficacious and affordable financially for commoners.
6. Cues to the manoeuvrability of macrophage function to be a game changer
Macrophages are capable of integrating an enormous and impressive amount of information regarding the identity and virulence of pathogens, as well as endogenous cues present in their microenvironment, in order to modulate the immune response to best protect the host. Over 30 years of extensive studies that advanced our understanding that the NF-kB signalling module consists of five NF-kB monomers (RelA/p65, RelB, cRel, NF-kB1 p50 and NF-kB2 p52) which can dimerize to form up to 15 unique transcription factors and interact with the kB consensus motif found in many gene promoters, as well as five inhibitory proteins (IkBα, β, ε, γ and δ) that make up the IkB protein family. What the author is trying to focus on is that so many genes involved in the functional manoeuvrability of the macrophage have been deciphered but any control or more precisely in the domain of therapeutic regulatory aspects are yet to be discussed, except the revelation of the
Negative regulation of NF-kB signalling was stated in an interesting mode. The synthesis of new IkB proteins or modules and subsequent reactivation of the pathway can lead to a periodic oscillation of active NF-kB translocation between the nucleus and cytosol. The newly synthesized IkB proteins bind to active NF-kB dimmers and remove from DNA binding and shuttling back to the cytoplasm where the complex can be reactivated and IkB can again be ubiquitinated and degraded via the proteasome. This is a powerful negative feedback loop. The balance of positive and negative feedback signals has a profound impact on the transcriptional outcome of NF-kB activation. Recent studies showed that immune cells challenged with traditional immune stimuli showed substantial and significant variations in the NF-kB response dynamics in the different contexts of at different times in the cell cycle. The positive feedback and sustained NF-kB nuclear occupancy was also proposed and described as dose-dependently induced by LPS action. Recurrent NF-kB oscillations between the cytosol and nucleus are linked to gene expression and studied at the single-cell level to decipher the transcription dynamics. But in case of an individual cell and its effect it is segregated, the body system during illness undergoes multi-cellular cross-talk based events. The NF-kB activation, transcription dynamics and gene expression studies in a single cell might result in better outcome of multiple NF-kB, IkB and IKK genes functioning to monitor or perturb or to better investigate how they can be controlled to interact between macrophages and other immune cells. Central to this ability, there are ways in which NF-kB signalling is modulated based on shifting thresholds of activation, capacity of various classes of the PRR to integrate information acquired and keeping over all tight regulation of transcription through rigorous positive and negative feedback loops. Fitting of these components together in the diverse context and how we may be able to modulate or interfere with them to the benefit of patients is an important field of research as envisaged. In this context, the interference to the action of leishmanial lipids to the NF-kB expression control, the mechanisms involved thereby needs to be demonstrated in an experimental set-up. But so long the beneficial aspects could be exploited and if it is proved to be satisfactory then therapeutic application would be well come. It is therefore imperative how we can harness the benefit of the leishmanial lipids or the preparations based on the use of the whole promastigote. The reader may go through the review [19] for a better and more detailed understanding.
The lipid(s) like the ones isolated from attenuated strain showed no toxicity at a dose of 500 mg per kg of body weight and no body weight loss treating at a dose 50 mg per kg. body weight in mice. The macrophage cell, RAW264 stimulated by LPS (bacterial endotoxin) showed ROS production and enhancement of active phagocytic uptake but upon treatment with leishmanial lipid both the stimulatory responses were reduced to more than half.
7. Antimicrobial resistance, attempted mitigation, Leishmania promastigote nonspecific host defence
Antibiotic resistance has become one of the greatest threats in human health care set-ups dealing with the issues for successful prevention and treatment of persistent infections. Misuse and overuses of antibiotics including in the agriculture sectors and allied fields have made a tremendous impact in the field of antibiotic resistance development. But spontaneity of environmental evolution, enormous mutational ability of bacteria and the capacity for passing the resistant genes through horizontal gene transfer system created significant impending factors to antimicrobial resistance mitigation. Multifactorial threats of antimicrobial resistance have posed numerous complex issues affecting countries across the globe. However, three categories have come out remarkably as patients, health care and economics to name them succinctly. At present, ‘Stewardship’ has been objectively imposed to get out of the problem to the rescue in the situation.
Israel, one of the most advanced countries in the world, whose health care system run wholly by the state, imposed the best possible ‘Stewardship’ in 2007–2008 by the introduction of containment for the country-wide outbreak of antibiotic-resistant
There was another study that reported that the faecal, oral and skin bacterial microbiome and antibiotic resistome of the members of an isolated Yanomami Amerindian village in Venezuela were analysed with the revelation of very interesting observations. Their ancestors arrived in South America more than 11,000 years ago and had no known exposure to antibiotics till at the time of this investigation. In their microbiome, they carry bacteria that harbour functional antibiotic resistance genes, including those that confer resistance to synthetic antibiotics [21]. Thus, it appears that functional AR genes occur as a feature of human microbiome even in the absence of exposure to commercial antibiotics. Thus, overexposure to or misuse of antibiotics does not essentially poise the dangers of antibiotic resistance though it is a factor no doubt.
The report described in 1992 that most of the bacteria associated as a contaminant with
In another study in Iran, among the 84 (patients) studied, 65 (77.4%) had a positive culture of bacteria in the
Lastly in the laboratory of the author, the mice were primed with attenuated
The questions of anti-immunology, one of the fundamentals, need to be addressed in the view of AMR (antimicrobial resistance). The pathogenic bacteria, virus and even fungal cellular constituent conventionally exert their anti-immunology strategies through their first encounter with the host macrophage after invasion. Here, the surface molecules of the interacting species, the macrophages and the invading agents must have direct molecular encounter. The antigenic/pathogenic component of the infectious agent having been masked with the promastigote-derived molecules supposedly, would surely be blocked to interact with the host cell (macrophages presumably). The pathogen would have very limited scope to make open the strategic options to compromise with the host cell. Again within 2–3 days, if there is any opportunity the pathogen could have to interact, a fresh injection (progressively) with a higher dose would result in the contact between the leishmanial constituents and host intracellular components along with antigens. By that time, their effective co-localization would result through fusion of late phagosome and lysosomal entities within macrphages. The lipoprotein acting as the pro-inflammatory cytokine inducer or lipids of the promastigotes acting as anti-inflammatory cytokines producing agents might have a regulatory role. For example,
8. Leishmanial promastigote Sphingolipids, apoptosis, and cancer
Attenuated leishmanial sphingolipids induce apoptosis in Sarcoma 180 cancer cells through the regulation of tumour development
As the theme of this chapter is the macrophage as game changer in the future treatment paradigm, the author invented a method for drug delivery system using IgG (intravenous fluid) and a schematic representation of the process has been given inFigure 1 [26] along with Figures 2 and 3. Figures 2 and 3 described the experience-based therapy or therapeutic vaccine developed by
All the products are injectable and contained protodin (IBL) as base material. All the information is given on good faith and may need proper clinical evaluation at user end but an expert clinical trial conducting physician valued these items as highly contributory (personal communication).
9. Conclusion
The two figures are the representations of Figures 2 and 3, displaying the attenuated
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Immuno Biological Laboratories or IBL ( nic name Dr. Ray’s Lab.) (1954-2005—the period of regulatory authority approved therapeutic vaccine producer and marketing firm with Valid Drug Licence issued by Govt.), P-91, Lake Road, Calcitta,-29 , India. (owned by Late Aruna Roy, Proprietress and wife of Late Dr. J.C. Ray M.D., the founder director of Indian Institute of Chemical Biology, currently one of the Prime Biomedical Research organization of India)