Evaluation of Modulators of cAMP-Response in Terms of Their Impact on Cell Cycle and Mitochondrial Activity of Leishmania donovani.

With the identification of novel cAMP binding effector molecules in Trypanosoma, the role of cAMP in kinetoplastida parasites gained an intriguing breakthrough. Despite earlier demonstrations of the role of cAMP in the survival of Leishmania during macrophage infection, there is essential need to specifically clarify the involvement of cAMP in various cellular processes in the parasite. In this context, we sought to gain a comprehensive understanding of the effect of cAMP analogs and cAMP-cyclic nucleotide phosphodiesterase (PDE) inhibitors on proliferation of log phase parasites. Administration of both hydrolyzable (8-pCPT-cAMP) and nonhydrolyzable analogs (Sp-8-pCPT-cAMPS) of cAMP resulted in a significant decrease of Leishmania proliferation. Among the various PDE inhibitors, etazolate was found to be potently antiproliferative. BrdU cell proliferation and K/N/F-enumeration microscopic study revealed that both cAMP analogs and selective PDE inhibitors resulted in significant cell cycle arrest at G1 phase with reduced S-phase population. Furthermore, careful examination of the flagellar motility patterns revealed significantly reduced coordinated forward flagellar movement of the promastigotes with a concomitant decrease in cellular ATP levels. Alongside, 8-pCPT-cAMP and PDE inhibitors etazolate and trequinsin showed marked reduction in mitochondrial membrane potential. Treatment of etazolate at subcytotoxic concentration to infected macrophages significantly reduced parasite burden, and administration of etazolate to Leishmania-infected BALB/c mice showed reduced liver and spleen parasite burden. Collectively, these results imply involvement of cAMP in various crucial processes paving the avenue for developing potent antileishmanial agent.

Role of leishmanial acidocalcisomal pyrophosphatase in the cAMP homeostasis in phagolysosome conditions required for intra-macrophage survival.

Exposure of Leishmania donovani to macrophage phagolysosome conditions (PC) (37°C and pH 5.5) led to increased intracellular cAMP and cAMP-mediated responses, which help in intra-macrophage survival pre-requisite for infectivity. In the absence of typical orthologs for G-proteins and G-protein coupled receptors, we sought to study the precise mechanisms for positive modulation of cAMP production during exposure to PC. Amongst two promastigote-stage specific membrane bound receptor adenylate cyclases (LdRAC-A and LdRAC-B), LdRAC-A appeared to function as a major cAMP generator following PC exposure. Pyrophosphate (PPi), an energy storage compound as well as a by-product of cAMP biosynthesis by adenylate cyclise, was found to be decreased following PC exposure. This may be due to microtubule and microfilament-driven translocation of acidocalcisomes near plasma membrane vicinity with concomitant increase of acidocalcisome membrane pyrophosphatase (LdV-H+PPase) and acidocalcisomal soluble pyrophosphatase (LdVSP1). Episomal over-expression and conditional silencing demonstrated regulatory role of V-H+PPase on cAMP trigger and consequent induction of resistance to macrophage-derived pro-oxidants and parasite killing. Furthermore, immunofluorescence analysis revealed possible co-localization of LdV-H+PPase and LdRAC-A during PC exposure. Collectively, these results suggest that translocation of acidocalcisome in membrane vicinity functions as a trigger for LdRAC-A-driven cAMP generation through depletion of PPi pool by LdV-H+PPase.

Morbid obesity is an independent risk factor for postoperative renal dysfunction in young adults: a review of the American College of Surgeons National Surgical Quality Improvement Program database.

BACKGROUND: Little information exists on the acute effects of elective surgery on renal function. Our aim was to determine if obesity was an independent risk factor for postoperative renal complications (RCs). METHODS: A total of 119,142 patients aged 18 to 35 years with body mass index (BMI) ≥18 kg/m(2) obtained from American College of Surgeons National Surgical Quality Improvement Project (2005 to 2010) were classified into standard BMI categories. Association between BMI and preoperative estimated glomerular filtration rate (eGFR; calculated using modification of diet in renal disease formula) was analyzed. Postoperative changes in eGFR and RCs were measured. Multivariate regression analysis was performed adjusting for all variables. RESULTS: Postoperatively, there was a reduction in eGFR among the overweight (-3.4 mL/min/1.73 m(2), P < .001), obese class I (-3.9 mL/min/1.73 m(2), P = .001), and obese class II (-5.3 mL/min/1.73 m(2), P < .001). The odds of any postoperative RC was significantly higher in obese class III patients (odds ratio = 2.01 95% confidence interval 1.07 to 3.76, P = .029). CONCLUSIONS: Results seen in patients with BMI greater than 40 indicate that BMI can serve as an independent predictor of RCs.

A soluble phosphodiesterase in Leishmania donovani negatively regulates cAMP signaling by inhibiting protein kinase A through a two way process involving catalytic as well as non-catalytic sites.

Intracellular cAMP level and cAMP mediated responses are elevated when Leishmania are exposed to macrophage phagolysosome conditions (37 °C and pH 5.5). Phosphodiesterases play major role in cAMP regulation and in the present study we have cloned and characterized a 2.1 kb cytosolic isoform of phosphodiesterase from Leishmania donovani (LdPDED) which plays important role in cAMP homeostasis when the promastigotes are exposed to macrophage phagolysome conditions for converting to axenic amastigotes. Domain characterization suggested the presence of two pseudo-substrate sites similar to the ones present in the regulatory subunit of cAMP-dependent protein kinase A (PKA) and a putative PKA phosphorylation site at T(708) of C-terminus of LdPDED. Deletion constructs and site directed mutagenesis revealed the ability of LdPDED to interact with L. donovani PKA catalytic subunits (LdPKAC1 and LdPKAC2) resulting in inhibition of kinase activity in one hand and increase of phosphodiesterase activity through PKA mediated phosphorylation at putative phosphorylation site on the other hand. This study therefore identifies a unique phosphodiesterase in L. donovani which appears to regulate cAMP-dependent PKA signaling through a two way process.

Post herpes-zoster scar sarcoidosis with pulmonary involvement.

Cutaneous sarcoidosis presents with a wide range of clinical presentations. An uncommon cutaneous manifestation is infiltration of old cutaneous scars with non-caseating granulomas known as scar sarcoidosis. Most of the patients with this clinical entity have other systemic manifestations, particularly pulmonary changes. We report a case of a 50 years old man, presenting with cutaneous sarcoidosis overlying scars of healed herpes zoster.

Lymphangioma of the tongue: response to intralesional steroids.

A 10-year-old boy presented with lymphangioma involving the anterior two-thirds of the tongue. Excellent cosmetic and functional results were achieved with multiple intralesional steroid injections. Various treatment modalities advocated for lymphangioma of the tongue are discussed.