Case Report: An Atypical Case of Post-Kala-Azar Dermal Leishmaniasis with Ulcers and Verrucous Lesions: Clinical and Therapeutic Implications.

About 75% cases of post-kala-azar dermal leishmaniasis (PKDL) occur in India. Although the classic description of PKDL is the progression from initial hypopigmented macular lesions to papules to plaques and nodular lesions, atypical morphologies are also seen and are easily missed or misdiagnosed. We report a case of a 27-year-old man who presented to us with multiple acral ulcers and verrucous lesions for 5 years. A diagnosis of PKDL was made based on slit skin smear, histopathology, and quantitative polymerase chain reaction. The patient was given combination therapy with four doses of liposomal amphotericin B and miltefosine 50 mg twice daily for 45 days. In this report, we discuss unusual morphologies of PKDL, the pathway to the diagnosis, and the therapeutic options available along with their efficacy.

Mycobacterium fortuitum-induced ER-Mitochondrial calcium dynamics promotes calpain/caspase-12/caspase-9 mediated apoptosis in fish macrophages.

Mycobacterium fortuitum is a natural fish pathogen. It induces apoptosis in headkidney macrophages (HKM) of catfish, Clarias sp though the mechanism remains largely unknown. We observed M. fortuitum triggers calcium (Ca2+) insult in the sub-cellular compartments which elicits pro-apototic ER-stress factor CHOP. Alleviating ER-stress inhibited CHOP and attenuated HKM apoptosis implicating ER-stress in the pathogenesis of M. fortuitum. ER-stress promoted calpain activation and silencing the protease inhibited caspase-12 activation. The study documents the primal role of calpain/caspase-12 axis on caspase-9 activation in M. fortuitum-pathogenesis. Mobilization of Ca2+ from ER to mitochondria led to increased mitochondrial Ca2+ (Ca2+)m load,, mitochondrial permeability transition (MPT) pore opening, altered mitochondrial membrane potential (ΔΨm) and cytochrome c release eventually activating the caspase-9/-3 cascade. Ultra-structural studies revealed close apposition of ER and mitochondria and pre-treatment with (Ca2+)m-uniporter (MUP) blocker ruthenium red, reduced Ca2+ overload suggesting (Ca2+)m fluxes are MUP-driven and the ER-mitochondria tethering orchestrates the process. This is the first report implicating role of sub-cellular Ca2+ in the pathogenesis of M. fortuitum. We summarize, the dynamics of Ca2+ in sub-cellular compartments incites ER-stress and mitochondrial dysfunction, leading to activation of pro-apoptotic calpain/caspase-12/caspase-9 axis in M. fortuitum-infected HKM.

Fluoride exposure abates pro-inflammatory response and induces in vivo apoptosis rendering zebrafish (Danio rerio) susceptible to bacterial infections.

The present study describes the immunotoxic effect of chronic fluoride exposure on adult zebrafish (Danio rerio). Zebrafish were exposed to fluoride (71.12 mg/L; 1/10 LC50) for 30 d and the expression of selected genes studied. We observed significant elevation in the detoxification pathway gene cyp1a suggesting chronic exposure to non-lethal concentration of fluoride is indeed toxic to fish. Fluoride mediated pro-oxidative stress is implicated with the downregulation in superoxide dismutase 1 and 2 (sod1/2) genes. Fluoride affected DNA repair machinery by abrogating the expression of the DNA repair gene rad51 and growth arrest and DNA damage inducible beta a gene gadd45ba. The upregulated expression of casp3a coupled with altered Bcl-2 associated X protein/B-cell lymphoma 2 ratio (baxa/bcl2a) clearly suggested chronic fluoride exposure induced the apoptotic cascade in zebrafish. Fluoride-exposed zebrafish when challenged with non-lethal dose of fish pathogen A. hydrophila revealed gross histopathology in spleen, bacterial persistence and significant mortality. We report that fluoride interferes with system-level output of pro-inflammatory cytokines tumour necrosis factor-α, interleukin-1ß and interferon-γ, as a consequence, bacteria replicate efficiently causing significant fish mortality. We conclude, chronic fluoride exposure impairs the redox balance, affects DNA repair machinery with pro-apoptotic implications and suppresses pro-inflammatory cytokines expression abrogating host immunity to bacterial infections.

Inducible headkidney cytochrome P450 contributes to endosulfan immunotoxicity in walking catfish Clarias gariepinus.

The effect of endosulfan metabolites on fish immune system is not well known. It is also not clear whether endosulfan accumulates in fish immune organs and undergoes metabolic biotransformation in situ. In the present study we investigated the role of headkidney (HK), an important fish immune organ on endosulfan metabolism and the long term effects of endosulfan metabolites on the fish immune system. C. gariepinus (walking catfish) were exposed to 2.884ppb of endosulfan (1/10th LC50) for 30d followed by their maintenance in endosulfan-free water for 30d for recovery. Endosulfan induced time-dependent reduction in the HK somatic index and histo-pathological changes in renal and hemopoietic components of the organ. At cellular level, exposure to endosulfan led to death of HK leucocytes. Gas-liquid-chromatography documented the presence of both α- and ß-isomers of endosulfan along with the toxic metabolite endosulfan sulfate (ESS) in the HK of exposed fishes. We report that ß-endosulfan accumulates more readily in the HK. Depuration studies suggested the persistence of ESS in the HK. Enzyme-immunoassay and qPCR results demonstrated direct relationship between cytochrome P450 1A (CYP1A) expression and ESS levels in the HK. Pre-treatment of HKL with CYP1A specific inhibitor α-Naphthoflavone (ANF) led to reduction in CYP1A mRNA, protein levels, and inhibited ESS formation together implicating the role of CYP1A on endosulfan metabolism. When the exposed fish were transferred to endosulfan-free water ('recovered fish') it was observed that after 30d of recovery period the concentration of endosulfan and its metabolite in the HK were significantly reduced, compared to 30-d exposed fish. We also observed improvement in HK histo-architecture but no significant recovery in HKL number and viability. Collectively, our findings suggest that HK plays an important role in endosulfan metabolism. We propose that endosulfan induces the activation of CYP1A in HK which led to the generation of persistent metabolite, ESS, resulting in immunotoxicity.

Calcium and Superoxide-Mediated Pathways Converge to Induce Nitric Oxide-Dependent Apoptosis in Mycobacterium fortuitum-Infected Fish Macrophages.

Mycobacterium fortuitum causes 'mycobacteriosis' in wide range of hosts although the mechanisms remain largely unknown. Here we demonstrate the role of calcium (Ca+2)-signalling cascade on M. fortuitum-induced apoptosis in headkidney macrophages (HKM) of Clarias sp. M. fortuitum could trigger intracellular-Ca+2 influx leading to the activation of calmodulin (CaM), protein kinase C alpha (PKCα) and Calmodulin kinase II gamma (CaMKIIg). Gene silencing and inhibitor studies established the role of CaM in M. fortuitum pathogenesis. We noted that CaMKIIg activation is regulated by CaM as well as PKCα-dependent superoxide anions. This is altogether first report of oxidised CaMKIIg in mycobacterial infections. Our studies with targeted-siRNA and pharmacological inhibitors implicate CaMKIIg to be pro-apoptotic and critical for the activation of extra-cellular signal regulated kinase 1/2 (ERK1/2). Inhibiting the ERK1/2 pathway attenuated nitric oxide synthase 2 (NOS2)-induced nitric oxide (NO) production. Conversely, inhibiting the NOS2-NO axis by specific-siRNA and inhibitors down-regulated ERK1/2 activation suggesting the crosstalk between ERK1/2 and NO is essential for pathogenesis induced by the bacterium. Silencing the NOS2-NO axis enhanced intracellular bacterial survival and attenuated caspase-8 mediated activation of caspase-3 in the infected HKM. Our findings unveil hitherto unknown mechanism of M. fortuitum pathogenesis. We propose that M. fortuitum triggers intracellular Ca+2 elevations resulting in CaM activation and PKCα-mediated superoxide generation. The cascade converges in common pathway mediated by CaMKIIg resulting in the activation of ERK1/2-NOS2 axis. The crosstalk between ERK1/2 and NO shifts the balance in favour of caspase dependent apoptosis of M. fortuitum-infected HKM.

Role of calmodulin-calmodulin kinase II, cAMP/protein kinase A and ERK 1/2 on Aeromonas hydrophila-induced apoptosis of head kidney macrophages.

The role of calcium (Ca2+) and its dependent protease calpain in Aeromonas hydrophila-induced head kidney macrophage (HKM) apoptosis has been reported. Here, we report the pro-apoptotic involvement of calmodulin (CaM) and calmodulin kinase II gamma (CaMKIIg) in the process. We observed significant increase in CaM levels in A. hydrophila-infected HKM and the inhibitory role of BAPTA/AM, EGTA, nifedipine and verapamil suggested CaM elevation to be Ca2+-dependent. Our studies with CaM-specific siRNA and the CaM inhibitor calmidazolium chloride demonstrated CaM to be pro-apoptotic that initiated the downstream expression of CaMKIIg. Using the CaMKIIg-targeted siRNA, specific inhibitor KN-93 and its inactive structural analogue KN-92 we report CaM-CaMKIIg signalling to be critical for apoptosis of A. hydrophila-infected HKM. Inhibitor studies further suggested the role of calpain-2 in CaMKIIg expression. CaMK Kinase (CaMKK), the other CaM dependent kinase exhibited no role in A. hydrophila-induced HKM apoptosis. We report increased production of intracellular cAMP in infected HKM and our results with KN-93 or KN-92 implicate the role of CaMKIIg in cAMP production. Using siRNA to PKACA, the catalytic subunit of PKA, anti-PKACA antibody and H-89, the specific inhibitor for PKA we prove the pro-apoptotic involvement of cAMP/PKA pathway in the pathogenicity of A. hydrophila. Our inhibitor studies coupled with siRNA approach further implicated the role of cAMP/PKA in activation of extracellular signal-regulated kinase 1 and 2 (ERK 1/2). We conclude that the alteration in intracellular Ca2+ levels initiated by A. hydrophila activates CaM and calpain-2; both pathways converge on CaMKIIg which in turn induces cAMP/PKA mediated ERK 1/2 phosphorylation leading to caspase-3 mediated apoptosis of infected HKM.

Revisiting a protocol for extraction of mycobacterial lipids.

Determination of lipid content of any biological sample is essential for various kinds of studies related to pathogenicity and drug development. Thus, reliable methods for the quantitative extraction of lipids are of critical importance. The mycobacterial cell wall is largely composed of lipids. Commonly used methods to extract lipids, such as the Bligh and Dyer method or the Folch method, yield a low amount of lipids when applied to mycobacterial cells. This study presents an efficient modification of Chandramauli's method, a less known method developed at this institute earlier that is able to yield a considerably higher concentration of mycobacterial lipids.