key: cord-0428043-lu3r2cws
authors: Das, Bikul; Pathak, Lekhika; Gayan, Sukanya; Pal, Bidisha; Saikia, Parthajyoti; Baishya, Tutumoni; Das, Nihar Ranjan; Das, Rupam; Maral, Mallika; Mahanta, Ranjit; Bhuyan, Seema; Gautam, Pratibha; Talukdar, Joyeeta; Sandhya, Sorra; Kalita, Deepjyoti; Swami, Vijay; Das, Krishna Ram; Bora, Dayal Krishna; Ghora, Jagat; Pulu, Ista
title: Stem cell basis of a host driven transmission of antigen packed aerosols: a novel mechanism of natural vaccination for tuberculosis
date: 2020-11-16
journal: bioRxiv
DOI: 10.1101/2020.11.14.382572
sha: bfdbac488126dfd0aa61024234cc261633377005
doc_id: 428043
cord_uid: lu3r2cws

Natural vaccination against pathogens are known to be achieved by herd-immunity i.e. infected human host provide immunity to the community by spreading the pathogen. Whether, infected human hosts transmit vesicle packed aerosols of pathogen’s antigen for natural vaccination of the community has not yet been considered. We have explored a traditional healing method of aerosol-inoculation against small pox and tuberculosis in the Sualkuchi-Hajo cultural complex of Kamarupa, an ancient Indian region known for tantra-based healing and spirituality. In the aerosol-inoculation method against TB, selected persons with TB (later identified as smear negative TB subject) are encouraged to spread good nigudah in the community by Kirtan chanting; the good Nigudah are thought to be present within bad-nigudah or invisible krimis (tiny flesh eating living being mentioned in ancient India’s medicinal text Caraka Samhita and Atharva Veda). A 15-years of contact TB investigation study, as well as laboratory study of aerosol obtained from smear negative PTB (SN-PTB) subjects led to the identification of good Nigudah as extracellular vesicles (EVs) filled with Mtb-antigen ESAT-6. We then developed a mouse model of aerosol-inoculation using SN-PTB subject derived aerosol EVs, and identified Mtb infected mesenchymal stem cells (MSCs) of the lung as the putative source of the ESAT-6+ EVs. These Mtb infected MSCs reprogram to altruistic stem cell (ASC) phenotype, which then secrete ESAT-6+ EVs to the aerosols; healthy mice receiving the aerosol develop Mtb specific herd immunity. These results expedite our ongoing work on the innate defense mechanism of ASCs against pathogen, and provide a novel mechanism of natural vaccination, where the host extracts appropriate antigens from a pathogen, and then spread it in the community via aerosols.

Pulmonary tuberculosis (PTB) is a global health burden caused by a rod shaped bacteria, the Mycobacterium tuberculosis (Mtb). The bacteria spread through aerosol in the community and now infect more than 2 billion people, killing 2 million people yearly. The pathogen enters into the respiratory tract and initiates a host/pathogen interaction with macrophages, an immune cell capable of antigen-delivery and export to other immune cells. However, bacteria may hide away from immune cell for years in a dormant state; we found that Mtb hijacks bone marrow (BM) stem cells as their protective niche (1) (2) (3) , confirmed by others (4) (5) (6) (7) . Under favorable condition, dormant Mtb reactivates in the lung causing extensive tissue damage and cavity formation, leading to clinical diseases of PTB (8-10). Only in 60% of PTB subjects, bacteria can be detected in the sputum and these smear positive cases can be isolated and treated to prevent community transmission. Whereas the rest of PTB cases may continue to spread the pathogen as the bacteria can't be detected in the sputum even after performing culture or testing for bacterial DNA (11) . These smear negative PTB subjects are public health challenge especially in rural health set up, where many of them may escape diagnosis and treatment for years and thus, spread the pathogen in the community. The role of these smear negative PTB subjects in either spreading or preventing the disease in the community is not yet clearly known. While managing PTB subjects in rural India through KaviKrishna Telemedicine Care (12, 13) , we have uncovered an Indigenous Knowledge System (IKS) based aerosol-inoculation practice that may provide novel insight about the role of these smear negative PTB (SN-PTB) subjects in spreading herdimmunity, a key element of social medicine approach against the pathogen.

IKS is the knowledge system inherited by indigenous culture, and it operates at local level of interaction through tradition/culture, and exhibit emergent property i.e. capable of producing new knowledge through interaction, and adaption to local environments (14, 15) . IKS based tool has been applied to develop effective public health approaches (13, 14, 16) . One prominent and ancient example IKS based public health approach could be the JivaUpakarTantra (JUT), also known as Vedic Altruism, a nearly extinct knowledge system of ancient Kamrupa (North East India, part of Bangladhesh and West Bengal) cultural complex that survived in rural pockets of Sualkuchi-Hajo area of Assam, India (17) (supplementary note 1). Our ongoing research indicate that JUT might have been involved in the traditional treatment and bioethical management for small pox in the Sualkuchi-Hajo area during 16-19 th century, when traditional inoculation/spiritual healing practice were widely used in Bengal and Assam (18) (19) (20) (21) . In fact, smallpox inoculation practice may have spread to China and medieval Europe from India through Buddhist monasteries (22, 23) . Interestingly, our ongoing IKS related study suggest that the JivaUpakarTantra (JUT) based approach against smallpox was unique, as it was aerosolbased and the underlying principle was Nigudah Jiva, which are considered as the lowest form of life in Jainism (24) as well as ancient Kamarupa tantra (17) (Chapter 1.3 and 1.4, pages 6-19; Reference 17) . Accordingly, selected person having smallpox were encouraged by tantrik (traditional healers of ancient Kamarupa) to chant mantras in front of others to transmit their good Nigudahs to the community so that bad Nigudahs presumed to cause small pox can be prevented from spreading (17) (Chapter 1.4, pages 12-19; Reference 17). Interestingly, BD witnessed Jagat Ghora (JG)'s JUT-based aerosol-inoculation approach against tuberculosis (TB) disease during early years (1993) (1994) of clinical practice in Sualkuchi. JG, a tantric healer and practitioner of JUT asked selected subjects with pulmonary TB (PTB) to perform Kirtan chanting in Hatisatra namghar (a local temple where the idea of KaviKrishna laboratory was initially conceived) to transmit good nigudahs in the community. JG was supported by KRD (one of the co-authors), a poet-philosopher who wrote several poems based on JUT and contributed to revive the metaphysics of JUT (25) . Importantly, BD noted that most of the PTB cases managed by JG were smear-negative PTB cases. Obviously, we had direct conflict with Tantrik JG's approach on managing PTB with JUT based approach, as we feared that such practice might spread the pathogen in the community.

However, we also considered that dialogue with IKS based traditional healers might lead to the discovery of undiscovered public knowledge (UPK) (26) , which may contribute to advancing our understanding of PTB. Such dialogue may be conducted by qualitative research tool such as focused group discussion (FGD), an useful tool for health care practitioner to enhance care for patients and community (27, 28) .We have been applying this FGD based research tool in conservation research (29), as well as clinical care for cancer patients in rural area of Assam (13) . We also speculated that guided-interaction between IKS and modern medical practice may lead to the emergence of novel insight about disease pathology (30). Hence, we sought to utilize FGD to conceptualize good-Nigudah of aerosol-inoculation, and then apply modern laboratory-based methods to study the potential rationale for aerosolinoculation.

Thus, FGD led us to conduct a contact investigation study among JG's aerosol inoculation subjects, and the eventual identification of ESAT-6 rich extracellular vesicles in the aerosols of smear negative PTB (SN-PTB) subjects. Importantly, upon intranasal injection of aerosol-EVs of SN-PTB subjects, recipient mice developed immunity against a laboratory strain of Mtb. Moreover, broncho-alveolar lavage (BAL) fluid of these immunized mice exhibited the presence of Mtb infected CD45 negative cells having "altruistic stem cell" (ASC) phenotype, and capable of secreting ESAT-6 rich EVs. These mice actively secreted rich EVs in their aerosols in an ASC dependent manner, as the reduction of these cells by a small molecular inhibitor FM19G11 led to decrease of EV rich aerosol production. These data indicate the potential role of a newly identified innate ASC defense mechanism (31) in the export of antigen-rich EVs by aerosol for possible spread of herd immunity. We then demonstrated that viable but not culturable (VBNC) deep dormant Mtb isolated from smear-negative subjects could reprogram human MSCs to ESAT-6 EV secreting ASCs. Finally, western blot approached confirmed the presence of CD63 rich ESAT-6+ rich EVs mainly in the SN-PTB subjects who's sputum contain VBNC. In this manner, we were able to identify a group of SN-PTB subjects as the potential agents of JG's tantric healing practice to spread good-nigudah or ESAT-6+ EVs via aerosol-inoculation. This discovery may enable to develop vaccine candidates by further studying the innate ASC based defense mechanism and their putative role in herd immunity.

In ancient civilization-culture based nation states such as India and China, many centuries old Indigenous knowledge system (IKS) continue to thrive as undisclosed public knowledge (UPK) of public health importance (14, 30) . Body-fluid based inoculation was practiced in ancient China and India against smallpox, which may have encouraged the idea of vaccine development (32). However, aerosol-based inoculation was also practiced against small pox in ancient Kamarupa, India's northeastern zone bordering China, and home to tantric healing practices since ancient time (17) (Chapter 5.6, pages 222-231, Reference 17) . Re-discovering the metaphysics of such healing practices may enable us to develop innovative scientific hypothesis community against bad-nigudah spread via aerosol from other PTB subjects. In JUT, the body defense mechanism of good-nigudah is known as the "Nigudah-Ojaskshayamatva pravritiarthapati", which can be roughly translated as the hypothesis (pravriti-arthapati) of "Nigudahimmunity".

Although, the concept of Nigudah-immunity fascinated us, we feared that such an aerosol-inoculation approach would spread TB in the community. Therefore, during the FGD session, we suggested JG to not include smear positive PTB (SP-PTB) subjects as bacteria is present in sputum of SP-PTB subjects. Further, we helped JG to modify his alternative medical practice so that he would take only SN-PTB subjects for the aerosol inoculation practice. As a trade-off, we agreed to conduct a contact TB investigation study among the Kirtan chanting devotees. JG and KRD wanted us to use the result of the contact investigation study for potential modeling of good-nigudah as per JUT's method of "tarka" ( Figure 1B , Supplementary note 1), an investigative tool of ancient India's Nyaya school of philosophy (37). Thus, guidedinteraction between IKS and modern medical practitioner led to the emergence of an experimental design of a contact TB investigation study to test the "nigudahimmunity" hypothesis.

Examination of close family and social contact of index cases at the time of diagnosis is a valuable method of case-findings as well as to study the disease transmission process (38). We selected an index case of smear positive PTB; a 32 year old male living in south-western part of Sualkuchi. Many people being in contact with the index case were also attending Kirtan in local Namghar (temple), Iswar Sri SriHatisatra (https://www.facebook.com/hatisatra/?rf=823873277776650), a spiritual tradition based on Sri

Sankardeva's Vaishnava tradition (107) . For initial 5 years (1994-1999 Figure 3A & B) . This result, although preliminary, and of limited use to draw a conclusion (because of small study population) encouraged us to conduct another FGD session of "tarka" to model good-nigudah, as described below.

Deriving a scientific hypothesis from metaphysical concepts is challenging (40) but may lead to knowledge emergence, a network process of interaction among the practitioners of local indigenous knowledge and practical philosophy (14) . "Satvata tarka", the JUT approach of interaction is a Hindu metaphysical method of reasoning and performing this method may lead to knowledge emergence may lead to knowledge emergence ( Figure 4A , and Supplementary note 1), DKB and BD both engaged in a dialectic process to discuss two seemingly opposite interpretations of the contact-investigation study result: 1) As per BD: smear negative subjects might have spread bacteria as good nigudah to other subjects (41) through Kirtan chanting leading to spread of immunity against the pathogen (the herd immunity). 2) DKB: SN-PTB subjects might have spread good components of bacteria as good-nigudah through Kirtan-chanting leading to spread of immunity against the pathogen (Nigudah-immunity, Figure 1A ). In the FGD session, as detailed -PTB; n=10; table 1) with mild to moderate chronic obstructive lung disease   (COPD) (FEV1/FEV6 <0.3, table 1 ), a clinical condition that we observed among the smear negative PTB subjects that participated in the contact investigation study. In COPD, EVs can be detected in the aerosols (46). In these SN-PTB subjects, exhaled breath aerosol (EBA) was collected by a microspiromter ( Figure 4C ) that simultaneously assessed the COPD status of these individual (FEV1/FEV; table 1). The EBA samples were devoid of Mtb-DNA ( Figure 4D ). In a similar manner, we collected EBA of COPD (n=8) and SN-PTB subjects who completed anti-TB therapy (n=8); Figure 4D and Figure 4E ), thus confirming the EV isolation method by ultracentrifugation.

To test our hypothesis that subject with SN-PTB may export Mtb-antigen rich EVs in their aerosol, the isolated EVs were subjected to ELISA to detect CD63, a marker of EVs. For the candidate Mtb-antigen, we selected the early-secreted antigenic target 6 (ESAT-6) that has been previously detected in aerosols of PTB subjects (48). EBA of healthy adult (n=7) served as control. The results are given in Figure 5 A Figure 5B ). Thus, it appears that EVs of SN-PTB subjects are enriched in ESAT-6 antigen.

However, others and ours own experience suggest that ultracentrifugation method of EV isolation lead to co-precipitation of protein aggregates. Indeed, our TEM image of Figure 4E shows the potential presence of protein aggregate in the isolated EV sample. We therefore considered that ESAT-6 may also be present outside the EVs but co-precipitated during the ultracentrifugation process of EV isolation. To exclude this possibility, we obtained purified CD63+ EVs by immunomagnetic sorting technique from the 3 subjects showing heterogeneous levels of ESAT-6 protein (#1,#2,#7, Figure 5B ) followed by western blotting confirmation of ESAT-6 in the sorted CD63+EVs. For this purpose, we used the exhaled breath condensate (EBC) method of aerosol collection to obtain a better yield of EVs (49) needed for the immunomagnetic based EV separation method. As shown in Figure 5C , western blot showed (Table 1) .

Therefore, further tests are required to find out the immunogenic relevance of EVs of SN-PTB including its potential ability to activate the Nigudah-immunity ( Figure 1A and 4A). Figure 5D , which shows that 0.5 µg/ml EVs containing 50ng ESAT-6 (patient #2) induced marked activation of MHC II and CD11c, whereas a similar dose of patient #7 derived EVs failed to activate DC. These results suggest the potency of selected SN-PTB subject derived aerosol-EVs (henceforth known as AESN-PTB) to induce DC activation in vitro. The findings also predict the potential ability of 0.5 µg EVs of patient #2 to induce anti-TB immunity in mice. Indeed, intranasal administration of patient #2 derived 0.5 µg EVs in 20 µl PBS weekly for two weeks showed marked increase of IFN-gamma response in mice (red arrow, Figure 5E ), which is comparable to immunity exhibited by a mice model of dormant Mtb that we previously characterized (1) . Importantly, these EV injected mice (n=3 mice per patient) showed reactivity to Ag85, a potent Mtb antigen being released in the aerosol of PTB subjects (54). Similar results were obtained from the patient #3-6 derived EVs (having ESAT-6 absorbance value of >0.3; Figure 5B ) were able to induce immunity, whereas EVs of 4/10 SN-PTBs, as well as all the subjects of drug treated SN-PTBs failed to induce IFN-gamma response, probably because of the lack of sufficient amount of Mtb antigens in the EVs. Noted that the equal dose of EVs of COPD subjects did not exhibit IFN-gamma response by splenic cells, confirms that there is no nonspecific IFN gamma response by EV of SN-PTB subjects.

Thus, even minute amount of aerosol EVs of some of the SN-PTB subjects were capable of generating IFN-gamma response against Mtb specific antigens in mice, highlighting the relevance of EV mediated antigen-export mechanism. Importantly, these findings now facilitate

to develop a mouse model of nigudah-immunity as described below.

The JUT's model of Nigudah-immunity ( Figure 1A /4B) provides a unique concept on herd-immunity: the export of pathogen-free antigen packed in EVs by infected individuals to the rest of a community. We have conceptualized a unique experimental model in mice that simulate the proposed mechanism of aerosol inoculation induced Nigudah-immunity ( Figure 6A ). Briefly, we conceptualized that in the AESN-PTB immunized mice, immune cells such as macrophages will actively produce ESAT-6 rich EVs (good-nigudah) by extracting the antigen from intracellular Mtb (bad-nigudah), as per the metaphysical modeling of good-nigudah ( Figure 4B ).

These good-nigudah carrying macrophages will reach the broncho alveolar lavagae (BAL) fluid, and then into the aerosol during coughing/chanting/sneezing in human, and induced-sneezing in mice. Macrophages are the candidate host cells, as these cells are being found to secrete Mtbantigen carrying EVs (55), but also the predominant cells in BAL fluid (56), a potential source for EVs in the aerosol. Additionally, MSCs, a newly identified host cells for Mtb intracellular dormancy and reactivation (1, 31) may also be a source of Ag-rich EVs, as these cells are well known for their EV secreting abilities (57, 58). Thus, we designed an experimental plan to look for Ag rich EVs in the BAL fluid, as well as in the aerosol of AESN-PTB immunized mice.

Three weeks after immunization, these animals were infected with a laboratory strain of Mtb to serve as the bad-nigudah. For this purpose, Mtb-m18b, an Mtb strain that we have extensively characterized in animal models (1, 2, 31) was used. This strain can be used in BSL-2 animal facility because of its low virulence and auxotrophic nature (59). Additionally, infection with this strain can be markedly reduced by prior immunization with BCG vaccine (59). Therefore, BCG immunized mice served as a control for immunization by AESN-PTB.

Thus, mice immunized with the AESN-PTB were infected with Mtb-m18b

(henceforth known as nigudah mice), and 8 weeks later (a required period for infection to establish in the lung), extraction of good-nigudah (Mtb-Ag rich EVs) from bad-nigudah (Mtb-m18b) was demonstrated by several assays. First, we demonstrated that healthy mice exposed to the aerosols of these nigudah mice exhibited ESAT-6 specific IFN-gamma response without being infected with Mtb-m18b. We noted that neither AE-COPD nor BCG immunized mice showed the ability to induce marked IFN-gamma response in the aerosol-exposed mice ( Figure   6B ) Taken together, the niguda mice (animal receiving AESN-PTB intranasal injection, Figure 5E , plus Mtb-m18b infection, Figure 6A ) exhibited a unique mechanism of herd immunity: export of ESAT-6 rich EVs in their aerosols for immunizing neighboring mice. The source of the ESAT-6 in the EVs is most likely the Mtb-18b, because, AESN-PTB injection alone did not cause the export of ESAT-6+ EVs (not shown). These results make this mouse model a putative model to study the proposed Nigudah-immunity.

The detection of CD45-cells in the BAL fluid of nigudah mice, and their potential role in extracting good-nigudah (Ag rich EVs) from bad-nigudah (Mtb) made us to consider the potential role of newly identified innate ASC defense mechanism in the Nigudah-immunity. Our ongoing work indicates that CD45-cells, which are mainly enriched in MSCs may exhibit an altruistic stem cell (ASC) defense mechanism against pathogen such as MHV-1 virus. Briefly,

ASCs exhibit an enhanced stemness phenotype as well as an altered p53/MDM2 oscillation to acquire a transient state of cytoprotective activity as a part of the innate stem cell defense mechanism for niche protection (2, 31, 53) . ASCs are dependent on hypoxia-induced transcription factors (53) which are involved in the exosome production in MSCs (62). Notably, ASC state facilitates Mtb replication, and therefore, could be potential source of Ag rich EVs.

Importantly, ASCs may be part of an unconventional immune response (63) against Mtb infection, as seen in BCG immunized mice challenged with Mtb infection (61, 64). Therefore, we considered to study whether Nigudah-immunity recruits the ASC defense mechanism for antigen-exports via aerosol.

To directly visualize Mtb harboring CD45-cells, and study their ASC phenotype in the Nigudah immunity model of mice, we used a GFP labeled Mtb-m18b strain (1) and recovered CD45-cells from the BAL fluid (8 weeks after Mtb-infection, as shown in Figure 6A ) for in vitro culture, including the status of intracellular Mtb-18b, and the putative ASC phenotype of the CD45-cells. A group of mice were treated with FM19G11, an inhibitor of ASC phenotype (53), and the animals were evaluated for the release of ESAT-6+ EVs in their aerosols and BAL fluid.

In this manner, we were able to directly visualize the Mtb-m18b intracellular to CD45-cells in the BAL fluid ( Figure 7A) . Notably, the CD45-cells appeared to exhibit a phenotyhpe of "enhanced stemness" ( Figure 7A ) that we recently showed to be important for dormant Mtb reactivation (31). Next, the intracellular bacteria number was higher than CFU counts suggesting the presence of VBNC ( Figure 7B ), which we confirmed by addition of early stationary phase supernatant (ESPSN) of Mtb-H37Ra, a conditioned media that can resuscitate viable but non culturable Mtb (65) . Then the real time PCR based gene expression data showed the ASC phenotype of CD45-cells, including the high expression of HIFs ( Figure Thus, human CD271+ BM-MSCs were infected with Mtb-S18b derived VBNC, and then we studied the ASC reprogramming by evaluating p53/MDM2 oscillation within a period of two weeks, and also the expression of stemness genes. The data is shown in Figure 8A -B, which clearly showed the VBNC-induced reprogramming of MSCs to ASC phenotype. Next, we found that VBNC but not actively growing m18b infected CD271+ BM-MSCs were capable of secreting ESAT-6+ EVs, without secreting live bacteria. Moreover, inhibition of HIFs by FM19G11 (12) led to marked reduction of ESAT+EV secretion by the VBNC infected MSCs ( Figure 8C -E). Thus, VBNC but not actively growing Mtb may acts as a source for ESAT-6+

EVs in MSCs, which needs further investigations.

After elucidating VBNC-Mtb as the source of ESAT-6+ EVs, we revisited our clinical data of Figure 5 , where the aerosols of 6/10 pretreated SN-PTB subjects exhibited ESAT-6+ EVs of greater than 0.3 absorbance at 450 nm, and also showed ability to induce the Nigudahimmunity in mice ( Figure 6 ). We speculated that these subjects might contain VBNC-Mtb in their BAL fluid, and therefore in the sputum. Indeed, whereas processed sputum of these subjects were negative for Mtb-DNA or culture, addition of ESPSN media in the processes sputum led to actively growing Mtb in 6/10 samples ( Figure 9A ). Thus, it appears that we have identified a group of SN-PTB subjects having VBNC-Mtb in sputum that spread aerosol EVs containing ESAT-6 into the community. The SN-PTB post treatment subjects without VBNC-Mtb (n=8) served as control. As a positive control for western blot, we used the EVs released by VBNC-Mtb containing ASCs (Figure 8 ), which clearly showed the presence of ESAT-6 as well as CD63 proteins ( Figure 9B ). Thus, AESN-PTB having VBNC-Mtb in the sputum contain ESAT-6+ EVs in their aerosols. Importantly, these EVs injected nigudah-mice ( Figure 6A ) showed 15-fold higher number of ASCs in the BAL fluid than the mice injected with the AESN-PTB having no VBNC-Mtb in their sputum (p<0.0001, Figure 9C ). Moreover, in these nigudah-mice, we observed a direct correlation between the number of ASCs in BAL fluid, and the amount of ESAT-6+ in the aerosol-EVs (p<0.019, Figure 9C ). These results indicate that VBNC versus non-VBNC group of AESN-PTB is capable of inducing a higher degree of innate ASC defense in the nigudah mice. Our results also show that the innate ASC defense is directly correlated with the Nigudah-immunity i.e. higher release of Ag rich EVs in the aerosols of these mice for exerting herd immunity.

Pulmonary tuberculosis (PTB) is not yet eradicated from the globe despite decades of TB control program (68) . Mtb, the causative agent of PTB enters into the human host via aerosol and causes wide-spread infection in the community. In this context, while in a densely populated country like India half of the community is infected with Mtb, yet only in 5-10% infected people develops PTB (69). It is not yet clear, how the 90% of population escape from PTB. In this manuscript, we reveal an IKS based approach of aerosol inoculation in the community to prevent TB infection, and its associated metaphysics known as Nigudah-immunity ( Figure 1A ). We carefully integrated this healing metaphysics to design innovative experimental plan, and found evidence for a novel mechanism of natural vaccination against TB. In this mechanism, the "Nigudah immunity" as described in Figure 9D Mtb to persist (71) . In contrast, our findings suggest that although MSC serves as a host for dormant Mtb (1) or MSC reprograms to ASC during dMtb reactivation (31), MSCs also induces an innate defense response characterized by herd immunity i.e. antigen export via EVs for vaccination of contact individuals.

Our study is unique in scientific approach, as we have integrated the metaphysics of a traditional indigenous healing concept, the Nigudah-Ojaskshayamatva or Nigudah-immunity ( Figure 1A ) to derive our experimental hypothesis on EV mediated antigen-export as a part of immune defense against a pathogen ( Figure 6A , and 9C). In this manner, we set an example that the integration of IKS, with modern science may lead to knowledge emergence, an idea that we proposed (14) , and working on to discover UPK remain undisclosed/near extinct in rural India.

We suggest that the Nigudah-theory of immunity may be the remnant of India's thousands years of tradition of variolation or inoculation/vaccination against small pox, as mentioned in the Sanskrit text "Sacteya" (32). Interestingly, the skillful application of Kirtan-chanting to spread good-Nigudah into the community may be effective in aerosol inoculation, as recent studies indicate that words including voice plosive consonants emit more aerosol particles from human respiratory tract during speech than words with voiceless fricatives (72) . This finding directly correlate with the more aerosol emission during Kirtan chanting as it does such a way, where a constant voice plosive words are played for a longer duration. In this respect, the results of our contact investigation study indicate apparent protection, although it is a small study without enough power to indicate significant protection. However, the efficacy of such antigen-export by aerosols to provide herd immunity remains to be proven. One key challenge is the potential very low amount of antigen that can be delivered through aerosols. Whether this small amount of antigen will be sufficient enough to induce effective immunity need to be investigated.

A previous study showed Mtb-antigen export during coughing (69) suggesting that aerosol may spread not only the pathogen but also antigens in the community. Additionally, aerosols may also contain potentially lipoprotein mixtures that may suppress immunity, as Mtb was found to use lipoproteins as a decoy mechanism to escape from macrophage response by inhibiting antigen processing and presentation (73, 74) . In this context, we found that mouse receiving aerosol-EVs were able to develop immunity, when injected intranasally, and or mouse to mouse aerosol inhalation for two weeks. Hence, it is possible that antigens delivered via EVs may be effective to mount a significant immune response. Infact, Growing bodies of research suggest the important role of EVs in the initiation and maintenance of adaptive immunity (42, 43). EVs are bioactive vesicles formed either as microvesicles (100 nm to >1 uM), exosomes (30-150 nm) or ectosomes (100-500 nm). The role of EVs in adaptive immunity was first noted in three elegant papers published in 1996-1998, where exosomes derived from B cells were found to be enriched for MHC class II, as well as co stimulatory molecules like CD86 and could present antigens to sensitized T cells (75) (76) (77) , and carriage of tumor antigens to promote antigenspecific T-cell activation (78) . Subsequently, gut epithelial cells derived exosomes were found to carry MHC class II/peptides, and involved in dendritic cell mediated immune response (79, 80) .

Furthermore, exosomes containing viral RNA of infected cells were found to modulate innate immune response (81). In TB, the role of exosomes in cellular immunity has been discovered. whereas BD represented the modern medicine side (two circles of tarka, Figure 4A , and supplementary note 1). BD started the debate with an initial enumeration (udeshya): the devotee who was chanting Kirtan did not suffer from PTB, because they were infected by the bacteria;

there body developed immunity against the pathogen. DKB identified the element of anistaprasangayukti (reasoning based on unwanted consequence) in BD's presumption. DKB stated that our conclusion that devotee may be infected by the pathogen is an unwanted effect of BD's initial observation that smear-negative subjects do not show bacteria in sputum; without bacteria in their sputum, how could they spread the pathogen to others? BD replied that 30% of smearnegative subjects convert to smear-positive subjects as the disease progress. So, it is possible that 3 of the smear-negative chanting devotees eventually became smear positive and infected the rest, who received low-dose of bacteria enough to trigger immunity to resist bacterial growth in lung. Those who did not attend Kirtan did not get infected with a low dose bacterium, and therefore, when they were exposed to high dose bacteria by smear-positive people in the community, their immunity was not strong enough to resist the bacterial growth in the lung.

However, DKB reached an alternative explanation by applying the purvavat-anuman method, which is the unperceived effect from a perceived cause (37)(page-32). DKB inferred that goodnigudahs spreading from the smear-negative subjects during Kirtan chanting protected the rest of the devotees from TB. To this explanation, BD applied samasya-laksana (doubtful feature) and stated that good-nigudah is an imaginary thing, and therefore, cannot be a factor for reasoning.

To this argument, DKB applied satavata-anuman (inference based on prior knowledge), and Smear negative subjects were selected as described previously (1, 67) and in the result section. were prepared (asked to take rest for 2-3 hours before the procedure, drink enough liquid, and then do not take any food/drink an hour before the procedure), and asked to exhale into a microspirometer after a deep breath so that their forced expiratory volume can reach the FEV1 (forced expiratory volume in one second)>2 liters and PEF (peak expiratory flow) of > 300 liters/minute.

They were then asked to exhale by mouth into the spirometer mouthpiece for every 3-5 seconds.

The breathing process was done for 10 minutes. The procedure was well tolerated by all the participants. The spirometer was taken into the laboratory for the aerosol collection, and processing for the isolation of extracellular vesicles (EVs) (details are explained in the method section for extracellular vesicle isolation). The aerosol samples (suspended in 0.1% BSA) were examined for amylase activity (#ab102523, Abcam; detection limit of 0.2 umol/l) and found to contain <1 umol/l amylase suggesting minimal contamination by saliva. The protein content in the aerosol was measured by Bedford assay (micro assay; detection limit of >1 μg /ml); we found average 10.4 ± 4.2μg/ml protein per 15 minutes of procedure, out of which, 2.5 ± 1.4 μg/ml were EVs. To increase the EV yield and also make the sample saliva free, we have used the exhaled breath condensate (EBC) method of aerosol collection previously used to isolate EVs in subjects with COPD (49). Thus, in selected subjects (where ESAT-6 EVs was detected following initial screening, and required enough EVs for in vivo mouse experiments), a home based EBC collection method was used. Briefly, subjects were prepared 3-4 hours before the procedure, and asked to exhale into a drinking glass (250 ml size). The drinking glass was soaked in ice cold 0.2% BSA, remaining BSA was discarded, and then the glass was wrapped in a frozen gel pack.

Study subjects were asked to exhale into the glass for 15-20 minutes; frozen gel pack was changed periodically 4-5 times to keep the glass ice cold. EBC (approximately 1.5 ml containing 20.5 ± 6.3 μg/ml protein, out of which, on average 5 μg/ml was EVs; no amylase was detected suggesting minimal contamination with saliva) was collected and immediately subjected for EV separation (described below). The procedure was repeated 3-4 times weekly for 2-3 weeks to obtain enough EV materials (50 μg/subject containing >10μg of ESAT-6, enough doses to immunize 5 mice) for the mouse experiments ( Figure 6-7) . Then, collected EVs were concentrated in a 0.5 ml aliquot in PBS containing 0.1% BSA, and stored at -80C for long-term storage for in the in vivo use as previously described (100). Sputum samples were also collected from these subjects over three consecutive days, and then transported to Ranbaxy lab, Mumbai product. This CD63+ sorted EVs were subjected to western blotting to confirm the presence of ESAT-6 antigen. As EBC derived aerosols showed high and better yield of EVs than EBA, EBC was performed for all the study subjects and EVs were isolated from EBC derived aerosols by ultracentrifugation and stored at -80 for future animal studies.

The EVs were also isolated from the aerosols of immunized Mtb-m18b infected mice (n=5 for each of the four subjects). Briefly, Mtb-m18b infected immunized mice (8 weeks after infection) were allowed to breath inside a 50ml tube for 5 minutes/day following sneezinginduction by intranasal injection of hypertonic saline (10 μl drop once that cause 4-5 sneeze/ 5 minutes). PBS was added in the tube where aerosols were collected from 5 mice (total 50 minutes of sneezing in two weeks). Aerosols were processed for ultrafiltration followed by ultracentrifugation to obtain EVs as described above. Approximately ~15 ug EVs were collected/5 mice/50 minutes of cumulative sneezing in two weeks.

The size distribution of EVs was measured on a ZetasizerNano 

The procedure was performed as described previously (12) . The EVs pellet suspended in RIPA was used to perform western blot to check the expression of ESAT-6 (anti-ESAT-6 antibody (#ab26246, AbcamInc, USA) and CD63 (#NBP2-42225SS, Novus

Biologicals, Centennial, CO, USA). immunized mice were selected for m18b Mtb infection and herd immunity mice model, as their IFN response against ESAT-6 was >1.5ng/ml, a value that can resist bacterial invasion (59).

We tested whether immunization with aerosol EVs served as a protective mechanism in mice against a M. tuberculosis challenge as described earlier (82) subjects. Also, we investigated, if the aerosol-mediated immunity against Mtb-m18b infection also involves antigen-export to neighbors, we developed a unique mouse model where aerosols from infected mice were allowed to be inhaled by healthy mice. The healthy mice were exposed to the aerosol of m18b-infected mice that received prior immunization either with BCG or Aerosol-EV of SN-PTB subjects. Briefly, the m18b infected mice (4 weeks after infection) were allowed to breath inside a 50ml tube for 5 minutes/day following sneezing-induction by intranasal injection of hypertonic saline (10 μl drop once that cause 4-5 sneeze/ 5 minutes);

immediately after, healthy mice were allowed to breath into the aerosol rich tube for 5 minutes/day. The procedure was repeated for two weeks, and then both infected mice and healthy mice were sacrificed. Then, the healthy infected mice were subjected to measure Mtb-CFUs in their BAL fluid, and whole lung to confirm the immunization against m18b infection. Mtb-CFU media was supplemented with 50µg/mg streptomycin as described (1).

Mtb-m18b-GFP bacteria were manually counted per microscopic field, and the data was used to with PBS-T at RT in the dark, and air-dried before scanning. Plates were scanned and analyzed using an Odyssey IR scanner using Odyssey imaging software 3.0. Contact investigation study was conducted among Kirtan chanting group exposed to smear negative and smear positive subjects. For initial 5 years (1994-1999 EBC products were pooled together from the 4/6 VBNC and 4/4 non-VBNC subjects, and EVs were isolated by density gradient centrifugation method. EVs (10 μg loading in each well) obtained from CD271+ ASCs infected with VBNC ( Figure 8E ) served as the positive control. C.

Correlation between the number of CD45-ASCs cells in the BAL fluid, and the optical density reading of the ELISA assay for ESAT-6 in the aerosol-EVs (5 μg/ml) of the corresponding mice.

Data is obtained from the experimental results of Figure 6C&D . D. A schematic model of EVs efficiently deliver the Mtb antigen to DC and other antigen-presenting cells leading to T-cell priming. In the subjects with active PTB, EV-induced immunity may also activate the innate ASC defense mechanism (31) including the extraction of Mtb-antigen from the VBNC, and export antigen rich EVs into the BAL fluid.

IKS is the knowledge system inherited by indigenous culture, and it operates at local level of interaction through tradition/culture, and exhibit emergent property i.e. capable of producing new knowledge through interaction, and adaption to local environments (14, 15) .

As depicted in Figure 1 , JUT has a unique metaphysics of medical treatment. The metaphysics is based on an Ayurvedic concept of "Vyadhikshamatwa" (equivalent to immune defense mechanism) (34-36) that body defense mechanism of a TB subject can extract good-nigudah (mentioned in ancient Hindu and Jain sacred texts) from bad-nigudah (pathogen itself as per JUT-UPK) of PTB subjects and spread these extracted good-nigudha via aerosol inoculation during Kirtan chanting to devotees. Then, this good-nigudah provides protection to the community against bad-nigudah spread via aerosol from other PTB subjects. In JUT, the body defense mechanism of good-nigudah is known as the "Nigudah-Ojaskshayamatva pravritiarthapati", which can be roughly translated as the hypothesis (pravriti-arthapati) of "Nigudahimmunity". The immunity activates a subtle layer, the Avatar Kosha (Altruistic layer of subtle body), and this is why the treatment method is known as Jiva Uapakar (Biological altruism).

Since the healing method has its root in the Vedic concept of Avatar (around the cultural complex of Hayagriva Temple of Hajo, Kamarupa), hence, it is also known as Vedic Altruism (Chapter 1-3; Reference 17). This idea of Avatar Kosha is based on the Satvata tarka element as described below.

JUT uses a tarka method as a part of the knowledge emergence. "Tarka" (suppositional reasoning) is a well-established investigation method for new knowledge generation in ancient

Indian philosophical system (37). Tarka emphasizes on the social dimension of knowledge, where resolving controversy is reined by reasoning rather than the sources of knowledge.

Paradigmatically, tarka is called for in order to establish a presumption of truth in favor of one thesis against a rival thesis, both of which are supported by a putative source of knowledge. The thesis and the counter thesis both are backed up by apparent knowledge sources such as genuine inferences (the most common situation) or competing perceptual or testimonial evidences. One scholar tries to refute the reasoning of the competing scholar and try to establish the knowledge validity of his/her own thesis by identifying weakness in opponent's thesis. These weaknesses include anista-prasanga (unacceptable consequences) or breaks in intellectual norms in the rival's knowledge. It is presumed that such tarka event, which can be compared to modern world peer-review concept, may enhance the overall knowledge of both the groups leading to increase of overall knowledge generation in the society including the knowledge-emergence process of IKS (14) .

The tarka based reasoning spread throughout ancient India, and was known as Anviksiki during the composition of Nyaya Sutra by Gotama (500 BC), and then probably spread throughout India (97, (110) (111) (112) as evident by the presence of Tarka culture in Kamarupa (present day Assam). The In the Satvata tarka process, one such idea/circle engage in argument with an opposite idea/circle through a structured process, where each of the idea go through "parnima" (transformation) to sharpen each other's "yukti" (reasoning). This process of dynamic interaction, "satvata sanjog" between the two opposite ideas lead to "prajna agan" i.e. knowledge emergence, a component of IKS (14) ; Figure 3 contact investigation study (Figure 1-3 

As depicted in Figure 1 in this note, we were able to formulate the metaphysics behind JG's good-nigudah based aerosol inoculation against PTB. The epistemology of this metaphysics, the "satvatatarka" or dialectic method of reasoning is based on Figure 2 Parniamvada concept has similarity with Western dialectic (113) .

After completion of the 15 years of initial contact investigation study (1994-2009), we did the second Satvata tarka session to discuss the study results. Dayal Krishna Bora (DKB), an eminent vedic scholar agreed to represent JG and KRD, as both of them passed away. DKB was familiar with JG and KRD's Satvata tarka method, as the trios were involved in researching on Jiva Upakar Tantra (page 53, 428-530, Reference 98). Thus, DKB represented the good Nigodahypothesis side, whereas BD represented the modern Immunology side (two circles of tarka, Figure 2A ). The tarka started with initial enumeration (udeshya): to find out why chanting showed apparent protection from PTB. Then, the tarka process started with the postulation (pravritti-arthapati) stage. BD argued from the Immunology point of view (the Immunology circle) that apparent protection was due to latent Mtb infection acquired during chanting; there body developed immunity against the pathogen. This idea was opposed by DKB, who provided argument based on Jiva Upakar Tantra (JUT): the apparent protection was due to spread of good nigoda. Then, DKB moved to the Yukti-tarka circle, and identified the element of anistaprasanga yukti (reasoning based on unwanted consequence) in BD's presumption. DKB stated that BD's conclusion that devotee may be infected by the pathogen is an unwanted effect of BD's initial observation that SN-PTB subjects do not show bacteria in sputum; without bacteria in their sputum, how could they spread the pathogen to others? BD replied that 30% of SN-PTB subjects convert to smear-positive subjects as the disease progress. So, it is possible that 3 of the smear-negative chanting devotees of the study eventually became smear positive and infected the rest, who received low-dose of bacteria enough to trigger immunity to resist bacterial growth in lung. Those who did not attend Kirtan did not get infected with a low dose bacterium, and therefore, when they were exposed to high dose bacteria by smear-positive people in the community, they developed PTB. However, DKB reached an alternative explanation by applying the purvavat-anuman (unperceived effect from a perceived cause) (37,97). DKB inferred that good nigoda spreading from the SN-PTB subjects during Kirtan chanting protected the rest of the devotees from TB by inducing immunity. To this explanation, BD applied samasya-laksana (doubtful feature) and instated that good nigoda is an imaginary thing, and therefore, cannot be a factor for reasoning. To this argument, DKB applied satavata-anuman (inference based on prior knowledge) to conceptualize the physical feature of a good-nigoda.

DKB referred to an ancient law of duality (dvaita-vedanta), where it is possible for good to emerge from bad through the process of protibimbavada (law of reflection). The theory was proposed by an 8 th century vedantic scholar Padmapada that certain effects may be the mirror image of a cause (110) . DKB asked BD to draw the general features (laksana) of the TB causing bacteria in a piece of paper. BD drew a seed (DNA and cytoplasm) inside a sack (plasma

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JUT) or Vedic altruism based Satvata tarka, and Prajna Agan (Knowledge emergence) Background: Jiva Upakar Tantra or Vedic Altruism

JUT) is an a traditional medical text of ancient Kamarupa (Sualkuchi-Hajo cultural complex located 25 miles north east of Guwahati, the main city of

Avatar layer), a transient subtle body that emerges in our body during sickness, similar to the emergence of Avatara such as Fish Avatar during the Great Flood. Because, Avatar acts altruistically (Upakara in Sanskrit), hence, KRD translated Jiva Upakar Tantra as Vedic Altruism

the lead author of this paper did research on JUT both in Assam and also adjacent Bhutan between 1995-1998. BD found JUT to be a form of Indigenous Knowledge System, and borrowed its philosophical idea, Prajna Agan (equivalent to western idea of Knowledge emergence) to develop a concept of cultural emergence (Das B. Globalization and Emerging Opportunities of Indigenous Cultures

According to KRD, the Satvata tarka was practiced in Hatisatra temple, and had five circles (pancha-cakra) or stages of debate

Inference based on prior knowledge (satvata/purvavat-anuman)

Suppositional reasoning (yukti-tarka)

Special feature of doubt (samasya-laksana) and 5. Examination of a new idea

Some of these five elements were part of an ancient system of knowledge generation through reasoning (page 2 and 32, reference 37), whereas the other elements are the product of local transformation of knowledge system as predicted by local adaptation of indigenous knowledge system

These five elements or circles of tarka are initiated by two foundational element of tarka (Figure 2): 1. Activity based postulation (pravrtti-arthapati) and 2. Active experience (Vijana)

These seven concentric circles are represented by one big idea/view/circle (Figure 2), and known as guru-asana vyuha (the concentric seat of a guru)

DKB then applied the law of reflection to draw a similar sack with part of the seed inside, and termed it as good-nigoda. DKB then invoked the observation of a desire (ichha-prataskhya) and cited the example of Dadhicchi (an ancient sage who donated his own bone for greater cause of social benefit) to infer that some good chemical exist in the bacteria, which will be present in the seed of good-nigoda. DKB then asked BD to come up with any known features by medical science that can resemble the image of good-nigoda (Figure 4B, main text). To this question, BD replied that bacteria-infected cells of our body might be able to capture some antigens from the bacteria

DKB then invoked the observation of a desire (ichha-prataskhya) and postulation (arthapati) elements of the tarka to insist that goodnigoda can be observed in the aerosol of smear-negative subjects. Both DKR and BD then invoked the last element of the tarka, pariksha-dharana or the examination of a new idea, and concluded that prajna-agan or knowledge emergence happened, as we obtained an experimental idea to directly test the existence of good-nigoda in aerosols. Thus, we drew the image of the good-nigoda (Figure 4B, main text), and hypothesize that an extracellular vesicle-containing antigen derived from Mtb may serve as the model for good nigoda

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