Macrophage as the Game Changer of the (Future) Therapeutic Paradigm

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.

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 ‘The Doctor’s Dilemma’ (1906).The human host and other mammals are continuously exposed to the environment along with numerous microorganisms, including disease-causing dreadful infectious pathogens. Before the advent of the age of antibiotics, human host defended against the infection with his own in-built immune system or through its modulation and, possibly traditionally experience-based useful therapeutic agents. The miraculous success of the penicillin and other antibiotics to combat the bacterial pathogens primarily made an impact of historical significance. More so when the dominant dogma ruled the world that diseases are originated essentially from a germ. If the germs are not identified, would be identified in future course of time. Pasteur’s discovery of vaccine and diseases of especially bacterial pathogen originated ones have been combated profoundly by useful and effective antibiotics. So the clinical world was on the verge of declaring that war against infection has been won. But slowly at a creeping mode, the pathogens developed strategies to defeat the assault of antibiotics and what it is now is an emerging threat of antibiotic-resistant pathogens. So it is time to pay attention to the old guard ‘the host own immune-mediated defence’ for the betterment of health care or the mode of treatment paradigm.

An attenuated Leishmania donovani or L. donovani (UR-6MHOM/1978) was maintained over decades in the institute, Indian Institute of Chemical Biology, where the author joined as an independent investigator way back in the 80s of the last century. The protozoan parasite being attenuated lacks disease-producing capacity. It can infect host cell macrophage but in the next generation, it ceased to survive. The author out of curiosity planned to see whether the promastigote as a heat-killed preparation would be capable to modulate the immune response of the host. He administered it into healthy normal BALB/c mice to monitor the changes that might take place in the host immune system. He chose bacterial LPS or endotoxin to induce shock in the host as the host macrophage is the target to interact with both the LPS and the promastigote. After 3 days of priming the mice could withstand lethal blow of LPS, indicating that the macrophage had a real change in the responsiveness. After 1 month of priming, the mice showed the development of delayed type of hypersensitivity (DTH) indicating macrophages not only acquired protecting ability against LPS but also had undergone fundamental functional changes that included the changes in T-cell repertories. The mice displayed antibody raising ability against an antigen when the promastigote was used as an adjuvant [1]. The principal effectors of DTH reactions are Th1 lymphocytes mediated events where macrophages function as APC. The antibody raising capacity of a host acquired by B-lymphocyte maturation and APC mediated antigen presentation along with helper Th2 cells in case of protein antigen. Therefore, the priming of the mice with the promastigote of attenuated Leishmania had a profound effect on the overall functional changes of the macrophages, to a larger extent in the entire immune system. It must be mentioned that attenuated promastigotes of Leishmania have been tried for vaccine development of mammals as a vaccine candidate. Over the decades, it has been extensively tried, but no adversity was reported though no acceptable vaccine candidate fulfilled all the criteria to make one a successful vaccine. It was however not explored to develop any therapeutic item or an adjunct therapy exploring the attenuated promastigote till then.

1.1 The attenuated leishmania promastigote and watery diarrhoea control

The bacteria E. coli colonize the infant gastrointestinal tract within hours of life, and thereafter, it remains harmless usually deriving mutual benefit with the host. But the barrier of the gastrointestinal tract may be breached under conditions of immune suppression or other debilitating attacks resulting in damage or leakage. Then, it can cause infection even being harmless ‘non-pathogenic’. Several distinct diarrheal syndromes accompanying the bacterial infection including diarrhoeagenic E. coli infection are very often faced by clinicians. One such strain was available in our laboratory and the organism caused diarrhoea in mouse when injected through the intraperitoneal (i.p.) route. The animal succumbs within 3–4 days after the infection. The infected animal showed profuse watery diarrhoea. It is documented that Enteropathogenic E. coli (EPEC) infection rapidly modulates electrolyte transport and induce watery diarrhoea [2]. The hallmark of this bacterial pathogenic infection is the development of an A/E (adhesion/effacing) lesion in the intestinal lumen. The Payer’s patch (PP), lamina propria (LPr) like immune organs lay as an embedded component below the mucosal surface constituted by the enterocyte monolayer and play a very important role in the regulation of immune response to resolve injury such as A/E lesion. LPr macrophages participate in the sampling of luminal content, via extension of transepithelial dendrites and phagocytose transgressing pathogens. By priming the mice with the attenuated Leishmania promastigote and after 2–3 days they were infected with this diarrhoeagenic bacterial pathogen. These animals instead of developing lethal diarrhoeal assault and dying found unaffected and protected completely [3]. The mechanism remained to be elucidated but constitutive IL-10, anti-inflammatory cytokine production by the intestinal macrophage and apoptosis induction could be proposed and that might have an impact in this case.

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 Leishmania promastigote or promastigote at first with immediate encounter causing the release of pro-inflammatory cytokines. Gradually, it alters to M2 phenotype, especially when promastigotes after being degraded release its constituent lipids, proteins etc. along with resultant anti-inflammatory cytokine release. So the resolution of enteropathogenic E. coli infection-mediated damage could be assumed due to the therapeutic aspects of macrophage response modification towards homeostasis attainment [4, 5].

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 in vitro system. The designed preparations were IC (intact cell), CH (cell homogenate), CD (cell debris) and CS (cell soup obtained after CD collection by centrifugation). Mouse peritoneal macrophage, human monocyte–macrophage, a permanent murine cell line macrophage J.774 and mouse splenocytes were the test cells. TNF-α, LPS, BCG and an immune suppressor betamethasone were selected as the test compounds or specimen as well documented macrophage response modifier.

The parasite Leishmania has two important ligands, gp63 and LPG (lipophosphoglycan) to interact with host macrophage but the LPG moiety was devoid of this strain. Only its gp63 moiety as a well-recognized ligand to interact with the host macrophage was present. Macrophage MR (mannose receptor) is presumably the most efficient among others such as CR1, CR3 and fibronectin receptors for its response modifying interaction.

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-E. coli (Green Fluorescence Protein expressing E. coli) bacteria. But such phagocytic up take by CH pre-exposed macrophages was slightly higher. In respect of antibiotic or drug resistance or even multidrug resistant clinical isolates of both Gram-positive and Gram-negative bacterial pathogens, no marked discriminatory action was noted apparently.

The in vivo study was conducted taking live animals (mice) such as BALB/c, Swiss, CBA/j and a wild strain. They all were primed with most of the promastigote preparations and after 5 days of priming challenged with lethal doses of E. coli^R (drug resistant), S. aureus^R (drug resistant) and E. coli K-13 (the diarrhoeagenic strain). The unprimed and carrageenan treated groups were also challenged in parallel. The differences in survival rate between the two groups, primed and unprimed was remarkable as the primed groups showed considerable improvement (80–30% or 50% higher). The preparation CH was used for priming of BALB/c mice mainly with multiple (seven arbitrarily chosen) doses at an interval of 4–5 days, and then challenged with diarrhoeagenic E. coli K13. It is not known how far the attenuated leishmania promastigote lipoprotein(s) relates to bacterial outer membrane protein. Whether it functionally correlated and arguably induce protection for a long time against the bacterial infections remained as a guess. But the effect of multiple doses priming of mouse with IC showed absolutely safe and acquirement of long-term protection. One of the interesting findings was that the spleen cells of multiple doses of CH primed mouse when cultured in the presence of CH or CH coupled E. coli K-13, the culture soup demonstrated the presence of IL-2, and confirming Th1 cell-mediated immunity or CMI development. The protective ability is acquired even by priming with one or two doses. Multiple doses and the frequencies of administration with respect to time gap made a remarkable difference in the protection profiles, namely using CH, IC and lipoprotein (LP), but a safe gap of 3 days is desirable between two injections of the promastigote preparation [6]. The impact of phagosome maturation and over all immune response modulation by the host is possibly most critical in view of macrophage as a game changer, and now IC, the intact promastigote of the attenuated Leishmania donovani (UR-6MHOM/1978)-mediated phagosome maturation would be dealt in details.

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 Leishmania was described for the first time implicating the role of macrophage to act as infection eradicating agent. The maturation indeed undergoes, a full range of changes and it progresses through subsequent modification out of sequential fusion with endosomes when the microbe already is inside the macrophage.

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 Leishmania promastigotes. Here described possibly the more documented evidence. The parasite Leishmania ensures their survival by inhibiting macrophage phagolysosomal fusion and so maturation is imperfect through a complex enzymatic process and reorganization of cytoskeleton structure, accumulating peri-phagosomal actin and reducing the recruitment of late phagosomal marker Rab7 and delaying recruitment of LAMP1 probably. Other strategies include modulation of macrophage for lower NO generation, reduced ROS, IL-12 and TNF-α production, NF-kB and AP-1 activation. The resolution of microbial infection, in contrast, depends on the arms of inflammatory response but at the later stage, it necessitates the induction of the anti-inflammatory cytokines such as IL-10, TGF-β and IL-4, the Th2 type cytokines. Activated protein kinase (MAPK) plays also a critical role because activation of p-44/42 MAPK leads to IL-10 production and p38 MAPK downregulation results in abrogated IL-12 production. This is now clear that within the same macrophage phagocytosis of microbial agents assigned to the destruction through the association with pro-inflammatory macrophage effector function and its apoptotic cell resolution requires anti-inflammatory or silent absorption. These are the characteristic feature of pathogenic Leishmania to establish infection, but its counterpart attenuated Leishmania strain was reported as a booster of respiratory burst and TNF-α production [7]. In the latest study, it was reported that attenuated strain of leishmania simultaneously induces both the forms of the cytokines to accomplish the jobs.

The macrophage exposed to the heat-killed intact attenuated L. (L. stands for Leishmania) promastigotes showed augmentation of ROS and NO generation, the former develops in a steady state with a gradual increase up to 24 hrs. to reach the peak while NO production was also time-dependent but noticed at 24 hrs. Both the species were highly microbicidal in action the attributes to combat the infection with bacterial pathogens. Macrophage phagosome membrane-associated gp91 phox and iNOS or induced nitric oxide synthase are instrumental behind the ROS and NO generation As already mentioned, the phagocytic uptake was significantly enhanced by latex particles, which were really non-physiological but provider of an index to mark the extent of response. E. coli-GFP (green fluorescence protein) expressing one was used to confirm the enhancement firmly. The fascinating observation was the enhanced uptake of pathogenic Leishmania strain (AG 83), as it is known that such pathogenic parasite usually limits the uptake, thus the action of the attenuated strain surpasses or overpowered the influences of the pathogenic strain-mediated interference. So, the strength of phagocytic engulfment appeared more forceful. It is not known if the time gapped multiple doses priming under in vivo would be more effective or force bearing to combat infection. Upon pre-exposure to macrophage with the promastigote, the Th1 type cytokines level raised significantly, such as the level of IL-12, IL-1β, TNF-α and IL-6 were induced markedly. Expression of Th2 cytokine namely TGF-β was lowered but another anti-inflammatory cytokine IL-10 had a significant increase. It might be mentioned that phagocytosis of microbial pathogen and apoptotic cell when to take place in the same macrophage then the former is under the regulatory dictum of pro-inflammatory cytokine while the latter is under the aegis of anti-inflammatory cytokine. It tells us that the clearance of both the microbial infective agents and the apoptotic dead cell is mediated by the macrophage simultaneously. The same macrophages on account of attenuated L. promastigote activation carry out the desired functions efficiently.

The heightened level of the expression and translocation of NF-kB and c-Jun, the crucial transcription factors were evident due to the attenuated L. promastigote exposure to the macrophages. It provides clearly the effect of the promastigotes, making host responses towards better defence. Both the transcription factors are critical regulators of pro-inflammatory cytokine release. The flow cytometric analysis of the L. promastigote pre-exposed macrophage for 4 hrs, confirming the increased accumulation of NF-kBp65 and c-Jun in the nuclear extract of the activated cell. It means that the macrophage function as progress towards the late phagosome with the received cues from within and as the consequences of released constituents of the attenuated L. promastigote. For example, as it would be shown later in detail that lipids isolated from the promastigote decrease the accumulation of these nuclear transcription factors in the Synovial Fluid Mononuclear Cell (SFMCs) of RA patients implicating benefit to the patients.

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 Leishmania.

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 L. promastigote is highly efficient in achieving this feat. The internalized particulate matter both apoptotic cell and bacteria or any other microbial pathogen are regarded as garbage for clearance. They must be degraded and digested or defects in degradation have a number of consequences. For example, the lack of degradation (of DNA during apoptosis) or lysis of the apoptotic cell within macrophage results in autoimmune disease [9]. Neutrophil-mediated killing is hampered by the slow or inadequate phagosome maturation and certain pathogens seem to use the phagocytic machinery to enter and/or live within the host, all these have the better opportunity to survive in the context of knowing the various steps of cargo handling by macrophages. However, the attenuated L. promastigote exposure to macrophages caused its activity modulation to a great height. It might lead to the attainment of the homeostasis of immune system by way of providing optimum and therapeutic attributes.

The macrophages, as the pivotal host cells whose response modification through L. promastigote-mediated phagosome maturation, have been described elaborately. Properly time gapped multidose administration of this promastigote-derived material could lead to the repeated response modification of macrophages in sequences. It can act as a disease combating agent after attaining appropriately stimulated physiological state without damaging the host. The intact cell (promastigote) or its preparations were empirical but there is provision to use it for the disease cure. However, such macrophage activity changes could be manoeuvred using its identified individual component(s). In view of this total lipids and sphingolipids were isolated and examined for i) the potentiality of the compounds to treat human diseases in future and ii) the compatibility of their use in humans. The attenuated heat-killed promastigote is unique in the sense that it did not elicit any harmful consequences and its safety is assured through the vaccine trial.

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 Leishmania promastigote, the working material here. The lipids isolated by Bligh and Dyer [11] method showed six spots upon TLC analysis following standard technique.

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 in vitro and in vivo system, in the latter case using animal models.

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 L. promastigote treatment so its level was thought to be determined to have an overall picture. Then, the changes in the level of NF-kB p65 in the nuclear extract of the SFMCs were determined to substantiate the basis of the effect of the lipids for lowering of the release of proinflammatory cytokines. SFMCs were a heterogeneous cell population composed of primarily monocyte–macrophage, fibroblast and synovial cells whose hyperplasia was a characteristic feature of the disease RA. So apoptotic cell death of the viable SFMCs was also used as a parameter to monitor the other aspect of the leishmanial lipids action. Currently, therapeutic intervention includes the treatment of RA patients with biological such as infliximab or etanercept and those are highly clinically efficient for RA suggesting that the neutralization of the TNF-α is the primary target of the disease control. These treatments were also found to delay joint destruction. So the treatment is to be targeted to the suppression of TNF-α release rather than neutralization and simultaneously to decrease the NF-kB p65 level both in extent and translocation parameter by the lipids. A rat model of Rheumatoid Arthritis developed by collagen-adjuvant induced RA was also used in parallel to see effect of leishmanial lipids.

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 L. promastigote induced apoptosis of both mouse and human melanoma [13, 14]. In this case of RA patients, the total lipid-mediated apoptosis of SFMCs was nearly 70% and 22–25% at a dose of 100 μg and 50 μg /ml of total leishmanial lipids for 48 hrs. Treatment. It was reported that normal peripheral mononuclear cells obtained from healthy donors suffered no significant cytotoxicity at a dose of 100 μg/ml. It might be referred that normal melanocytes were not vulnerable to leishmanial sphingolipids unlike its cancer counterpart, melanoma. The leishmanial lipids acted probably as an apoptosis inducer in a wider spectrum of cells other than cancerous one. Cells functionally altered due to pathological changes found susceptible to apoptosis after being treated with the attenuated L. promastigote lipids, but normal cells did not suffer such cytotoxicity (unpublished observation). Because the dead SFMCs cells revealed all the signatures of the apoptosis; the activation of caspase-3 and -9 and Bax, DNA fragmentation, cytochrome c release along with the alteration of mitochondrial membrane potential and downregulation of Bcl-2 [13, 14]. All these observations indicated and supported strongly that leishmanial lipids are a powerful suppressor of cytokine expression in relation to RA pathogenesis. It was further interesting to note that the effect of the leishmanial lipids was probably at the nuclear induction level rather than an individual constituent level such as neutralization of TNF-α or TNF-α receptor blocker level or anti-proliferative action provider like methotrexate, a drug used for RA management.

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 in vitro studies have been illustrated here but for in vivo studies, rats were used to conduct the experimentation in an animal model. Here, RA was induced by collagen-adjuvant (FCA)-mediated procedure with confirmation of paw swelling in 14 days. The rat serum level of TNF-α, IL-1β, and NO produced was reduced after the treatment with the leishmanial lipids. The paw swelling was reduced on 16th days returning close to normal. But it was interesting to observe that normal healthy mouse not having induced AR if treated with leishmanial lipids showed a higher levels of serum TNF-α and IL-1β but not show any detrimental consequences, compared with control (lipid untreated). It was possibly due to enhanced nonspecific host defence [16].

5. Macrophage, lipids of attenuated Leishmania donovani promastigote, sepsis attenuation

The macrophage-mediated therapeutic applications based on the L. promastigote-derived bioactive molecules namely lipids, sphingolipids and lipoprotein and the intact promastigote itself have been described here briefly. As the clinical studies are far away from the concept and so organized studies are to be focused to compare some old experience-based therapies and a prospective experimental therapy.

5.1 The case of sepsis

Prospective experimental therapy vs. experience-based therapy (or therapeutic vaccine or vaccine in bed).

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 Escherichia coli remain as one of the most common pathogens (up to 60%) in intraperitoneal infections with high mortality rates. Moreover, the recognition of CD14-TLR4 complex by cell wall components of Gram-negative bacteria (E. coli) may lead to activation of the inflammatory responses. The overproduction of inflammatory cytokines generates systemic activation which affects vascular permeability and gives rise to metabolic changes that can lead to tissue injury and eventually to the failure of various major organs to induce mortality. Infectious inflammatory stimuli elicit acute lung distress which may be perceived as the most fatal cause affecting the initiation of various cellular cascades. It may also lead, firstly, to pre-eminence of inflammatory cells in the interstitium and alveolar spaces and, secondly, to an increase in PMN-derived proteases and oxidative metabolites in the bronchoalveolar lavage fluid (BALF). Local inflamed cells in the lung interstitium activate the pulmonary capillary endothelium culminating in the expression of adhesion molecules on the endothelial cell. Strategies aimed at preventing such cell activation, and thus attenuating systemic inflammation relevant to lung injury are proposed to control the sepsis.

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 E. coli challenged murine sepsis model. The inhibition of production of serum cytokines including TNF-α, IL-1β, IL-6, IL-12 and IL-17 and of the chemokine MIP-2, was achieved by administering LTL. In the in vitro culture of macrophages, such inhibition of the sepsis mediators by LTL was evidenced after E. coli induced stimulated release of the cytokines and chemokines. TNF-α and IL-1β are known as signature cytokines that initiate an acute inflammatory cascade to cause inflammatory injury leading to the recruitment of inflammatory cells to the affected organ. TNF-α a pleiotropic cytokine plays a central role to regulate other cytokines especially IL-17 and IL-12 that are rapidly generated in bacterial E. coli infection. LTL showed attenuation of the systemic inflammatory reactions and multiple organ failures associated with abdominal sepsis syndrome. It is well known that E. coli provokes the signalling through its receptor cluster involving CD14 and TLR4. The activation of the IκB kinase complex (IKK) led to phosphorylating the inhibitory IκB proteins that is necessary for ubiquitination and degradation leading to the release and subsequent translocation of NF-κB p65 from the nucleus. TNF-α and IL-1β, macrophage induced cytokines stimulate the production of a variety of chemokines, namely, macrophage-inflammatory protein-2 (MIP-2), this was attenuated by LTL together with functional effectors in the bronchoalveolar lavage fluid (BALF).

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 E. coli. And this was proposed by the observation that combating the inflammatory response to infection and exaggerated inflammation related to tissue injury have been successfully compromised. In the present case, lung injury was reduced by the suppression of the expression of macrophage cytokine-induced cell adhesion molecules. Thus, macrophage functional modulation improved the survivability with alteration of pathological changes in mice with septic mice lungs [17, 18].

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 Dr. J.C. Ray M.D. and became the first director, as a founder director of the renowned biomedical research institute of India, currently Indian Institute of Chemical Biology, Kolkata, India.

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 Immuno Biological Laboratories or IBL and the product is named ANASEPSIN (IBL) but technically Streptococcus and Staphylococcus Anti pyogenic Vaccine. Besides the two mentioned microorganisms, others included were Pneumococcus, E. coli (B. coli as described at that time) and Pseudomonas aeruginosa (pyocyaneus as described at the then time).

The documents were reached to the author in 2007, after 2 years of his retirement and Dr. J.C. Ray M.D. passed away in the late seventies (1975) of the last century. The author joined to Indian Institute of Chemical Biology in 1984 as an independent investigator. So, no scope of communication was possible with the great Scientist who saved lives over the decades. For not complying with the GMP norms, the unit remained closed since 2005, but few documents were made available to the author as stated.

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 Leishmania as per available information). The injection begins at 0.1 ml, then 0.2, 0.4, 0.6, 0.8 and 1 ml dose subcutaneously and progressively at an interval of 3 or 4 days. Then doses with higher count containing killed bacteria were used and time gapped injections were scheduled enhancing to 4–5 days interval.

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 Leishmania promastigote-mediated events and more defined the lipids of this parasite promastigote covered very recently. The ‘promastigote tricks’ were discovered and explored way back in the early 50s of the last century by Dr. J.C. Ray, M.D. founder director of present Indian Institute of Chemical Biology, Kolkata, India. The lack of publicity and scientific exposition might be the reasons other than meeting the fund crunch for his research endeavour Dr. Ray, kept the firm (IBL) in a kind of obscurity about his novel and great service as a developing giant of therapeutic vaccine. But recently, the disclosure of the lipids of the said promastigote and some scientific endeavour made it possible to indicate that the changes in functional aspects of the macrophage are possible to manoeuvre its activities during the NF-kB nuclear transcription factor expression dynamics. What is more pertinent is the non-haematopoietic cell of bone marrow origin possibly lung epithelial cell, after having been injured, was also found to have remedial fate during the leishmanial lipid’s exposure to it under in vivo system. The author has discussed the promastigote and lipids already and mentioned the attributes to the effect of leishmanial lipoproteins in host defence, especially protection against LPS threat. But the severe downregulation of the expression of LPS induced cytokine release in terms of TNF-α production and survival improvement after infecting the lipoprotein primed mice with the bacterial pathogens have been described as a phenomenon of host protection and an example of macrophage-mediated defence.

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 Klebsiella pneumonia (CRE—carbapenam-resistant Enetrobacteriaceae) in her 27 hospitals, as a nationally implemented intervention strategy. The incidence of nosocomial CRE was significantly reduced from 55.5 to 11.7 cases per 100,000 patient days [20]. But total prevention or mitigation of the problem of antibiotic resistance remains at a level of concern.

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 Leishmania major lesion was eliminated during parasite treatment. Only Klebsiella and E. coli were retained when treated even with paromomycin and were considered antibiotic-resistant contaminants. Surprisingly, total elimination of these antibiotic-resistant contaminants was evidenced only during the healing process and took 20 days following the completion of the parasite removal by treatment [22]. It implicated that as a consequence of parasite clearance the host immune system was altered and acquired the capacity of resolving the resistant bacteria.

In another study in Iran, among the 84 (patients) studied, 65 (77.4%) had a positive culture of bacteria in the L. major lesions indicating of secondary bacterial infection. The infections are usually treated with antibiotics but there were controversies about whether any positive gain of antibiotic treatment is rendered by antibiotics. Here, the treatment of cutaneous lesion with glucantine not only resolves the lesions but also cured the secondary infection in the presence or absence of antibiotics for selected groups. It clearly implicated that during the treatment and cure of cutaneous leishmaniasis the host immune response has been modulated and that helped the patients get rid of secondary bacterial infection even in the absence of antibiotics. It led to suggest that the secondary bacterial infection associated with lesions of cutaneous leishmaniasis was cleared (irrespective of sensitive or resistant varieties of bacterial pathogens) due to immunomodulation resulting upon curing the parasitic disease [23].

Lastly in the laboratory of the author, the mice were primed with attenuated Leishmania promastigote (heat-killed) preparations remained unaffected after 3 days of priming, if challenged with the pathogenic as well as drug-resistant clinical isolates [6].

Immuno Biological Laboratories or IBL described a curative vaccine with the heat-killed typhoid germs, namely Salmonella typhi., S. typhi A and S. typhi B in a colloidal preparation of attenuated Leishmania promastigote and claimed as an useful item for enteric fever treatment and sold for years (over 50 years). It was an excellent typhoid bacterial antigen mix capable of generating specific antibodies, stated by the product [IMMUNO-T.A.B(IBL) curative vaccine for the treatment of enteric fevers] literature. The company also described that the patients’ recovery was quicker and no relapse was noticed even though there were reports of chloramphenicol resistance in regular treatment with the antibiotic. This was probably the case because the phagosome maturation and innate immune response enhancing in the presence of promastigote-derived substances, all kinds of host-mediated events would be elicited towards enhanced pinocytosis, endocytosis and phagocytosis either sequentially or overlapping simultaneously. As the events are host-mediated and (heat-killed) Leishmania cell-mediated phenomenon where immune responses were never detrimental on the contrary it was to attain the condition of homeostasis upon curing.

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, Salmonella, an intracellular pathogen undergoes apoptosis in a macrophage with cell death, instead of escape when apoptosis is regulated by promastigote factors. There are two options of the host macrophage—either enhance the apoptosis in such a way that the pathogen gets involved in the apoptosis-mediated clearance and the other one is to face the detrimental phagocytic assault that the pathogen may suffer by degradation in a necrotic fashion. The promastigote sphingolipids are strong apoptosis inducers or powerful phagocytic assault creators by their capacity of proinflammatory cytokine/oxidative species release. So, it is apparently suggestive that the claim of the company, Immuno Biological Laboratories that no relapse of the Salmonella infection might be valid. The pathogen covered/sealed within the promastigote material could be engulfed by macrophages easily and such enclosed invader hardly has any opportunity to escape, but experimental demonstration demands to prove it.

8. Leishmanial promastigote Sphingolipids, apoptosis, and cancer

Attenuated leishmanial sphingolipids induce apoptosis in Sarcoma 180 cancer cells through the regulation of tumour development via angiogenic switchover. These bioactive leishmanial agents induce human A375 melanoma cell death via both caspase-dependent and independent cell death pathways or apoptosis. Both mouse and human melanoma in vitro displayed antineoplastic impact in tumour survival and heat-killed attenuated Leishmania promastigotes induces apoptosis of HepG2 cells through ROS-mediated p53-dependent mitochondrial pathway. The sphingolipids isolated from the attenuated promastigote Leishmania donovani (UR-6) indicated that their characteristic features might be to destroy the diseased cells of the host. For example, it induced apoptosis not only of cancerous but also of synovial fluid mononuclear cells of RA patients. But normal melanocytes and PMN do suffer any cytotoxicity upon exposure to the attenuated Leishmania promastigote or lipids, sphingolipids and lipoprotein derived from it while responding to functioning as expected. Once the apoptosis initiation is sensed, the macrophages come forward to clear the cargo within its sphere of activity. Apoptotic innate immune response is a state of immune-suppressive condition as the death is silent and the molecules in the fluid state of the promastigote preparation manipulate macrophage function through pinocytosis. Regarding melanoma destruction by apoptosis is an unique feature of Leishmania sphingolipids as there is no convenient way destined to eliminate these skin cancer cells. Intracutaneous injection starting from a very small dose, one can attempt to treat melanoma through apoptosis induction [24, 25].

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 Immuno Biological Laboratories and peer-reviewed published works based experimental therapies using the materials derived from attenuated as well as pathogenic Leishmania promastigotes characterized at molecular level in Figure 3 respectively. All schemes are given below.

Figure 1.

Macrophage mediated delivery of P, D or a through immunoglobulin encapsulated particles (future treatment paradigm using macrophage as the game changer – Hypothesis).

Figure 2.

Proposed treatment paradigm (experience based therapy – IBL evidence linked therapeutic items).

Figure 3.

Proposed treatment paradigm (experimental).

A glimpse of the products (used by people of India over 50 years) of a local company Immuno Biological Laboratories or IBL, Kolkata, India[27].

1. Immuno- T.A.B (curative) vaccine for treating enteric fever or typhoid. 2. Deneurin. A product developed by IBL that included dried substance of cobra venom and neurotropic bacteria. Dedicated to the treatment of cancer patients having been painful morbidity, a palliative therapy for cancer patient, here pain is compromised with snake venom, but no addiction developed like Morphin. 3. Dellergen. A product represents combination of specific and heterospecific hyposensitization antigens. Meant for treating the allergy and asthma. 4. Halsidin. Meant for anti-influenza and catarrhal vaccination. 5. Anasepsin. Already discussed for the treatment for sepsis. 6. Protodin (IBL). It is like the adjuvant or the non-specific vaccine can be used alone as an immunomodulator to enhance host immunity, even a patient receiving it at first visit to clinic would have no adversity, but could get time to diagnose properly. It can be used for boosting immunity in case a patient having milder symptoms or prior to COVID-19 infection as the author envisaged.

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 Leishmania promastigote-derived molecular constituents and the whole cell-based material-mediated macrophage activity modulation, respectively, towards its game changing parameter. It had been stated as self-explanatory styles with the emphasis of disclosing scientific basis by the peer-reviewed publications (Figure 3). This was only for the events not known prior to the experience-based therapeutic vaccine (Figure 2) that served people over more than 50 years. The third one or Figure 1 is a novel strategy envisaged by the author back in 2003 and provided with support for its experimental evidence. The explanation is omnipresent and interesting, as he did not find such an approach over the period since that time and desired exploration by the scientific world.