Developing Standard Treatment Workflows—way to universal healthcare in India

Primary healthcare caters to nearly 70% of the population in India and provides treatment for approximately 80–90% of common conditions. To achieve universal health coverage (UHC), the Indian healthcare system is gearing up by initiating several schemes such as National Health Protection Scheme, Ayushman Bharat, Nutrition Supplementation Schemes, and Inderdhanush Schemes. The healthcare delivery system is facing challenges such as irrational use of medicines, over- and under-diagnosis, high out-of-pocket expenditure, lack of targeted attention to preventive and promotive health services, and poor referral mechanisms. Healthcare providers are unable to keep pace with the volume of growing new scientific evidence and rising healthcare costs as the literature is not published at the same pace. In addition, there is a lack of common standard treatment guidelines, workflows, and reference manuals from the Government of India. Indian Council of Medical Research in collaboration with the National Health Authority, Govt. of India, and the WHO India country office has developed Standard Treatment Workflows (STWs) with the objective to be utilized at various levels of healthcare starting from primary to tertiary level care. A systematic approach was adopted to formulate the STWs. An advisory committee was constituted for planning and oversight of the process. Specialty experts' group for each specialty comprised of clinicians working at government and private medical colleges and hospitals. The expert groups prioritized the topics through extensive literature searches and meeting with different stakeholders. Then, the contents of each STW were finalized in the form of single-pager infographics. These STWs were further reviewed by an editorial committee before publication. Presently, 125 STWs pertaining to 23 specialties have been developed. It needs to be ensured that STWs are implemented effectively at all levels and ensure quality healthcare at an affordable cost as part of UHC

Reciprocity between Regulatory T Cells and Th17 Cells: Relevance to Polarized Immunity in Leprosy.

T cell defect is a common feature in lepromatous or borderline lepromatous leprosy (LL/BL) patients in contrast to tuberculoid or borderline tuberculoid type (TT/BT) patients. Tuberculoid leprosy is characterized by strong Th1-type cell response with localized lesions whereas lepromatous leprosy is hallmarked by its selective Mycobacterium leprae specific T cell anergy leading to disseminated and progressive disease. FoxP3+ Regulatory T cells (Treg) which are essential for maintaining peripheral tolerance, preventing autoimmune diseases and limiting chronic inflammatory diseases also dampen proinflammatory T cells that include T helper 17 (Th17) cells. This study is aimed at evaluating the role of Treg cells in influencing other effector T cells and its relationship with the cytokine polarized state in leprosy patients. Peripheral blood mononuclear cells from of BT/TT (n = 15) and BL/LL (n = 15) patients were stimulated with M. leprae antigen (WCL) in presence of golgi transport inhibitor monensin for FACS based intracellular cytokine estimation. The frequency of Treg cells showed >5-fold increase in BL/LL in comparison to BT/TT and healthy contacts. These cells produced suppressive cytokine, IL-10 in BL/LL as opposed to BT/TT (p = 0.0200) indicating their suppressive function. The frequency of Th17 cells (CD4, CD45RO, IL-17) was, however, higher in BT/TT. Significant negative correlation (r = -0.68, P = 0.03) was also found between IL-10 of Treg cells and IL-17+ T cells in BL/LL. Blocking IL-10/TGF-β restored the IL-17+ T cells in BL/LL patients. Simultaneously, presence of Th17 related cytokines (TGF-β, IL-6, IL-17 and IL-23) decreased the number of FoxP3+ Treg cells concomitantly increasing IL-17 producing CD4+ cells in lepromatous leprosy. Higher frequency of Programmed Death-1/PD-1+ Treg cells and its ligand, PDL-1 in antigen presenting cells (APCs) was found in BL/LL patients. Inhibition of this pathway led to rescue of IFN-γ and IL-17 producing T cells. Results indicate that Treg cells are largely responsible for the kind of immunosuppression observed in BL/LL patients. This study also proves that Treg cells are profoundly affected by the cytokine milieu and this property may be utilized for benefit of the host

Inverse Correlation between IL-10 and IL-17 in BL/LL.

<p>A) Treg derived high IL-10 negatively correlates with low IL-17+ CD4 T cells in BL/LL (n = 13) signifying polarized immunity in leprosy. B) No correlation was found in BT/TT (n = 13). Correlation was done using Spearman rank correlation coefficient; r value was equal to -0.65 while p value was significant at 0.01 in case of BL/LL.</p

T regulatory cells in Leprosy.

<p>A) Representative FACS plot showing enumeration of Treg cell frequency (CD4+, CD25+ and FoxP3+) in BT/TT vs. BL/LL patient B) Total frequency of circulating Treg (CD4+, CD25+ and FoxP3+) cells in PBMCs isolated from peripheral blood of BT/TT vs. BL/LL patients (n = 20) vs. Healthy Contacts (n = 10). C) Representative flow cytometric analysis showing IL-10 production following <i>in vitro</i> stimulation with <i>M</i>. <i>leprae</i> (WCL) vs. unstimulated for 48 h in a Leprosy patient with LL. D) Scatter Plots are showing total IL-10 production in gated CD4+ FoxP3+ cells in BT/TT vs. BL/LL (n = 15) vs. Healthy Contacts (n = 10). E) CCR4 surface expression on gated CD4+ FoxP3+ cells in different groups of Leprosy patients (BT/TT vs. BL/LL), (n = 10). Each dot represents a single individual. Median values are shown in each set while P value< 0.05 was considered to be significant. Total number of cells analyzed by flow cytometry were 500,000. Data analysis was performed with flowjo software. Statistical analysis was done using Student’s t test for unpaired samples.</p

Identification of Th17 cells in Leprosy.

<p>A) Representative flow cytometric analysis showing IL-17 production on gated CD4+ CD45RO+ memory T cells following <i>in vitro</i> stimulation with <i>M</i>. <i>leprae</i> (WCL) vs. unstimulated for 48 h in a Leprosy patient with BT. 6 h PMA stimulation was used as positive control. B) Scatter plots are showing cumulative IL-17 production in gated CD4+ CD45RO+ cells in different groups of Leprosy patients (BT/TT vs. BL/LL) and Healthy Contacts (n = 10). Each dot represents a single individual of BT/TT (n = 15) and BL/LL (n = 15) patient. C) Scatter graphs are showing CCR6 surface expression on gated CD4+CD45RO+ cells in different groups of Leprosy patients (BT/TT vs. BL/LL) (n = 10). Median values are shown in each set while P value< 0.05 was considered to be significant. Total number of cells analyzed by flow cytometry were 500,000. Data analysis was performed with flowjo software. Statistical analysis was done using Student’s t test for unpaired samples.</p

Effect of Th17 cytokines on FoxP3+Treg cells in BL/LL.

<p>A) PBMCs were cultured for 72 h in different conditions with or without the presence of cytokines inducing Th17 population such as TGF-β, IL-6, IL-23 and those secreted by Th17, IL-17 and IL-22 in different wells and readout was measured in terms of FoxP3+ on CD4+CD25+ cells on one hand and simultaneous IL-17 production by CD4+T cells. B) Bar graphs show the cumulative data in BL/LL (n = 5) of FoxP3+ expression on CD4+ CD25+ cells as against C) IL-17 production by CD4+T cells under the same conditions as mentioned above. Mean with SEM are shown in each bar while P value< 0.05 was considered to be significant. Total number of cells analyzed by flow cytometry were 500,000. Data analysis was performed with flowjo software. Statistical analysis was done using Student’s t test for unpaired samples.</p

IL-10/TGF-β suppresses Th17 in BL/LL.

<p>A) Flow cytometric analysis showing the increased production of IL-17 on total gated CD4+ T lymphocytes in a leprosy patient with LL under different conditions, Unstimulated vs. Stimulated with leprosy antigen only vs. Stimulated with antigen and blocked with antibodies against IL-10/TGF-β alone or in combination. B) Cumulative data of BL/LL (n = 5) patients are shown in the bar graphs with the same conditions as above. Readout was taken in terms of IL-17 by CD4+ T cells at the end of 72 h. Mean with SEM are shown in each bar while P value< 0.05 was considered to be significant. Total number of cells analyzed by flow cytometry were 500,000. Data analysis was performed with flowjo software. Statistical analysis was done using Student’s t test for unpaired samples.</p

PD-1/PDL-1 Interaction Suppress IFN-γ and IL-17 Effector cytokines in BL/LL.

<p>A) Percentage frequency of PD-1 expression on CD4+FoxP3+ Treg cells vs. B) non Treg cells, (CD4+FoxP3-) in BT/TT vs. BL/LL (n = 10) patients and healthy contacts (n = 10). Percentage frequency of PDL-1 expression on APCs, C) on CD14+ Monocytes D) on CD19+ B cells in BT/TT vs. BL/LL (n = 10) patients and healthy contacts (n = 10) in PBMCs isolated from peripheral blood. Each bar represents a single category. Error bars are shown in each set while P value< 0.05 was considered to be significant. Bar graph representation showing the increased production of E) IFN-γ and F) IL-17 on total gated CD4+ T lymphocytes in BL/LL patients of leprosy (n = 4). PBMCs isolated from peripheral blood were blocked with α-PD-1 or with α-PDL-1 monoclonal blocking antibodies separately or in combination and kept in culture for 72 hours with or without <i>M</i>. <i>leprae</i> WCL antigen. From cultured cells, T cells were analyzed for effector cytokine production by flow cytometry. Total number of cells analyzed by flow cytometry were 500,000. Each bar represents a single category. Mean with SEM are shown in each bar while P value< 0.05 was considered to be significant. Data analysis was performed with flowjo software. Statistical analysis was done using Student’s t test for unpaired samples.</p

Hair & skin derived progenitor cells: In search of a candidate cell for regenerative medicine

Background & objectives: Skin is an established tissue source for cell based therapy. The hair follicle has been introduced later as a tissue source for cell based therapy. The ease of tissue harvest and multipotent nature of the resident stem cells in skin and hair follicle has promoted basic and clinical research in this area. This study was conducted to evaluate skin stem cells (SSCs) and hair follicle stem cells (HFSCs) as candidate cells appropriate for neuronal and melanocyte lineage differentiation. Methods: In this study, SSCs and hair follicle stem cells (HFSCs) were expanded in vitro by explant culture method and were compared in terms of proliferative potential and stemness; differentiation potential into melanocytes and neuronal lineage. Results: SSCs were found to be more proliferative in comparison to HFSCs, however, telomerase activity was more in HFSCs in comparison to SSCs. Capacity to differentiate into two lineages of ectoderm origin (neuronal and melanocyte) was found to be different. HFSCs cells showed more propensities towards melanocyte lineage, whereas SSCs were more inclined towards neuronal lineage. Interpretation & conclusions: The study showed that SSCs had differential advantage over the HFSCs for neuronal cell differentiation, whereas, the HFSCs were better source for melanocytic differentiation