Mediation of antitumor activity by AZD4820 oncolytic vaccinia virus encoding IL-12.

Oncolytic viruses are engineered to selectively kill tumor cells and have demonstrated promising results in early-phase clinical trials. To further modulate the innate and adaptive immune system, we generated AZD4820, a vaccinia virus engineered to express interleukin-12 (IL-12), a potent cytokine involved in the activation of natural killer (NK) and T cells and the reprogramming of the tumor immune microenvironment. Testing in cultured human tumor cell lines demonstrated broad in vitro oncolytic activity and IL-12 transgene expression. A surrogate virus expressing murine IL-12 demonstrated antitumor activity in both MC38 and CT26 mouse syngeneic tumor models that responded poorly to immune checkpoint inhibition. In both models, AZD4820 significantly upregulated interferon-gamma (IFN-γ) relative to control mice treated with oncolytic vaccinia virus (VACV)-luciferase. In the CT26 study, 6 of 10 mice had a complete response after treatment with AZD4820 murine surrogate, whereas control VACV-luciferase-treated mice had 0 of 10 complete responders. AZD4820 treatment combined with anti-PD-L1 blocking antibody augmented tumor-specific T cell immunity relative to monotherapies. These findings suggest that vaccinia virus delivery of IL-12, combined with immune checkpoint blockade, elicits antitumor immunity in tumors that respond poorly to immune checkpoint inhibitors.

Clinico-Mycological Study of Onychomycosis in Indian Diabetic Patients.

Background: Onychomycosis (OM) is the most common nail disorder accounting for 40-50% of all onychopathies. Onychomycosis is caused by dermatophytes in majority, mostly Trichophyton (T.) rubrum followed by T. mentragrophytes var. interdigitale. However, there is a variation in the etiological profile with the subset of population, time, and geographical location. In immunocompromised hosts, non-dermatophytic molds (NDMs) and yeasts like Candida albicans and Candida parapsilosis are the main causative agents. Diabetes mellitus (DM) is a well-established risk factor for OM. Aim and Objectives: This study was conducted to determine the clinical and mycological characteristics of OM in diabetic patients and to evaluate the clinico-etiological correlation, if any. Materials and Methods: Three hundred consecutive diabetic patients were screened, of whom 102 (34%) patients were diagnosed with OM based on clinical, mycological, dermoscopic, and histological criteria. Results: Distal lateral subungual onychomycosis was the most common clinical variant seen in 80 (78.43%) patients. Fungal culture was positive in 57 (55.88%) of which NDMs constituted approximately half (47.61%) of the isolates, followed by Candida species (30.15%) and dermatophytes (22.22%). The clinico-mycological correlation was performed to look for the association of various fungi with the clinical type of OM. Distal lateral subungual onychomycosis was majorly caused by NDMs (51.02%), followed by Candida species (28.57%), and dermatophytes (20.40%). Conclusion: Non-dermatophytic molds are increasingly incriminated as the causative organisms for OM in DM and must be considered as potential pathogens in the present scenario, thus necessitating the change in the treatment options accordingly.

Inhibition of amyloid ß1-42 peptide aggregation by newly designed cyclometallated palladium complexes.

Uncontrolled amyloid aggregation is a frequent cause of neurodegenerative disorders such as prions and Alzheimer's disease (AD). As a result, many drug development approaches focus on evaluating novel molecules that can alter self-recognition pathways. Herein, we designed and synthesized the cyclometallated pyrene (Pd-1 and Pd-3) and anthracene (Pd-2) based palladium complexes ([Pd((L1)Cl] Pd-1, [Pd(L2)Cl](Pd-2), and [Pd(L3)Cl] (Pd-3)). This study explores the effect of these complexes on the aggregation, fibrillation, and amyloid formation of bovine serum albumin (BSA) and Aß1-42 peptide. Several spectroscopic methods were used to characterize all the Pd-complexes, and the molecular structure of Pd-3 was determined by X-ray crystallography. The secondary structures were studied using circular dichroism (CD) and transmission electron microscopy (TEM), while amyloid aggregation and inhibitory activities were investigated using the Thioflavin-T (ThT) fluorescence assay. Molecular docking of the Pd-complex (Pd-3) was done using fibril (PDB: 2BEG) and monomeric (PDB: 1IYT) peptides using Auto-dock Vina. As a result, the hydrogen bonding and hydrophobic interaction between the aromatic rings of the Pd-complexes and the amino acids of amyloid-ß peptides significantly reduced the production of ordered ß-sheets of amyloid fibrils and protein aggregation in the presence of Pd-2 and Pd-3 complexes.

Total synthesis, structure elucidation and expanded bioactivity of icosalide A: effect of lipophilicity and ester to amide substitution on its bioactivity.

The first total synthesis of icosalide A, an antibacterial depsipeptide that is unique in that it contains two lipophilic beta-hydroxy acids, has been achieved by following Fmoc solid-phase peptide synthesis in combination with solution-phase synthesis. The ambiguity in the absolute stereochemistry of icosalide A has been resolved by synthesizing the reported structures and other relevant diastereomers of icosalides and comparing their NMR data. NMR-based structure elucidation of icosalide A revealed a well-folded structure with cross-strand hydrogen bonds similar to the anti-parallel beta-sheet conformation in peptides and displayed a synergistic juxtaposition of the aliphatic sidechains. 12 analogues of icosalide A were synthesized by varying the constituent lipophilic beta-hydroxy acid residues, and their biological activities against Bacillus thuringiensis and Paenibacillus dendritiformis were explored. Most of these icosalide analogues showed an MIC of 12.5 µg mL-1 against both bacteria. Swarming inhibition by icosalides was least in B. thuringiensis (8.3%) compared to that in P. dendritiformis (33%). Furthermore, this is the first report of icosalides showing assured inhibitory action (MIC between 2 and 10 µg mL-1) against the active stage of Mycobacterium tuberculosis and cancer cell lines such as HeLa and ThP1. This study could help optimize icosalides for anti-TB, antibacterial, and anti-cancer activities.

Evolution of Mycobacterium abscessus in the human lung: Cumulative mutations and genomic rearrangement of porin genes in patient isolates.

BACKGROUND: Mycobacterium abscessus subspecies massiliense (M. massiliense) is increasingly recognized as an emerging bacterial pathogen, particularly in cystic fibrosis (CF) patients and CF centres' respiratory outbreaks. We characterized genomic and phenotypic changes in 15 serial isolates from two CF patients (1S and 2B) with chronic pulmonary M. massiliense infection leading to death, as well as four isolates from a CF centre outbreak in which patient 2B was the index case. RESULTS: Comparative genomic analysis revealed the mutations affecting growth rate, metabolism, transport, lipids (loss of glycopeptidolipids), antibiotic susceptibility (macrolides and aminoglycosides resistance), and virulence factors. Mutations in 23S rRNA, mmpL4, porin locus and tetR genes occurred in isolates from both CF patients. Interestingly, we identified two different spontaneous mutation events at the mycobacterial porin locus: a fusion of two tandem porin paralogs in patient 1S and a partial deletion of the first porin paralog in patient 2B. These genomic changes correlated with reduced porin protein expression, diminished 14C-glucose uptake, slower bacterial growth rates, and enhanced TNF-α induction in mycobacteria-infected THP-1 human cells. Porin gene complementation of porin mutants partly restored 14C-glucose uptake, growth rate and TNF-α levels to those of intact porin strains. CONCLUSIONS: We hypothesize that specific mutations accumulated and maintained over time in M. massiliense, including mutations shared among transmissible strains, collectively lead to more virulent, host adapted lineages in CF patients and other susceptible hosts.

Prevalence of onychomycosis in patients with diabetes mellitus: A cross-sectional study from a tertiary care hospital in North India.

Background Though diabetes mellitus (DM) is a well-recognised risk factor for onychomycosis (OM), the epidemiology of OM in diabetic patients remains largely unexplored, especially from the Indian subcontinent. Aims and objectives To estimate the prevalence of OM in diabetic patients, to identify and analyse risk factors, and correlate the severity of nail changes with glycemic control (HBA1c). Methods This cross-sectional, analytical study involved 300 diabetic patients. Patients with the clinical diagnosis of OM, supplanted by at least two of the four tests (KOH, culture, onychoscopy and nail histopathology) were considered cases of OM. Demographic and haematological profile was analysed using chi-square test/ Fischer's exact test. Logistic regression was applied to assess the independent risk factors. Results The prevalence of OM in DM patients was 34% (102/300) and significant risk factors included; age >60 years, male gender, closed shoes, disease duration >5 years, high BMI (>25) and lack of awareness about nail changes. Distal and lateral subungual OM (78%) was the commonest presentation followed by proximal subungual OM, superficial OM and total dystrophic OM. Correlation between HbA1c and the number of nails involved was found to be significant. Limitation As cases were recruited from a hospital setting, there could be chances of Berksonian bias. Conclusion The prevalence of OM in diabetic patients is high and the severity of nail changes correlates with HbA1C levels. It is important to diagnose OM early in order to treat and prevent complications.

Design and synthesis of piano-stool ruthenium(II) complexes and their studies on the inhibition of amyloid ß (1-42) peptide aggregation.

Misfolding and protein aggregation have been linked to numerous human neurodegenerative disorders such as Alzheimer's, prion, and Parkinson's diseases. Ruthenium (Ru) complexes have received considerable attention in studying protein aggregation due to their interesting photophysical and photo properties. In this study, we have synthesized the novel Ru complexes ([Ru(p-cymene)Cl(L-1)][PF6](Ru-1), and [Ru(p-cymene)Cl(L-2)][PF6](Ru-2)) and investigated their inhibitory activity against the bovine serum albumin (BSA) aggregation and the Aß1-42 peptides amyloid formation. Several spectroscopic methods were used to characterize these complexes, and the molecular structure of the complex was determined by X-ray crystallography. Amyloid aggregation and inhibition activities were examined using the Thioflavin-T (ThT) assay, and the secondary structures of the protein were analyzed by circular dichroism (CD) spectroscopy and transmission electron microscopy (TEM). The cell viability assay was carried out on the neuroblastoma cell line, revealing that the complex Ru-2 showed better protective effects against Aß1-42 peptide toxicity on neuro-2a cells than the complex Ru-1. Molecular docking studies elucidate the binding sites and interactions between the Ru-complexes and Aß1-42 peptides. The experimental studies revealed that these complexes significantly inhibited the BSA aggregation and Aß1-42 amyloid fibril formation at 1:3 and 1:1 molar concentrations, respectively. Antioxidant assays demonstrated that these complexes act as antioxidants, protecting from amyloid-induced oxidative stress. Molecular docking studies with the monomeric Aß1-42 (PDB: 1IYT) show hydrophobic interaction, and both complexes bind preferably in the central region of the peptide and coordinate with two binding sites of the peptide. Hence, we suggest that the Ru-based complexes could be applied as a potential agent in metallopharmaceutical research against Alzheimer's disease.

Safety and Efficacy of MEDI0457 plus Durvalumab in Patients with Human Papillomavirus-Associated Recurrent/Metastatic Head and Neck Squamous Cell Carcinoma.

PURPOSE: Tumoral programmed cell death ligand-1 (PD-L1) expression is common in human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC). We assessed whether a DNA vaccine targeting HPV-16/18 E6/E7 with IL12 adjuvant (MEDI0457) combined with the PD-L1 inhibitor durvalumab could enhance HPV-specific T-cell response and improve outcomes in recurrent/metastatic HPV-16/18-associated HNSCC. PATIENTS AND METHODS: In this phase Ib/IIa study, immunotherapy-naïve patients with ≥1 previous platinum-containing regimen (neoadjuvant/adjuvant therapy or for recurrent/metastatic disease) received MEDI0457 7 mg intramuscularly with electroporation on weeks 1, 3, 7, and 12, then every 8 weeks, plus durvalumab 1,500 mg intravenously on weeks 4, 8, and 12, then every 4 weeks, until confirmed progression and/or unacceptable toxicity. Coprimary objectives were safety and objective response rate (ORR; H0: ORR ≤ 15%); secondary objectives included 16-week disease control rate (DCR-16), overall survival (OS), and progression-free survival (PFS). RESULTS: Of 35 treated patients, 29 were response evaluable (confirmed HPV-associated disease; received both agents). ORR was 27.6% [95% confidence interval (CI), 12.7-47.2; four complete responses, four partial responses]; responses were independent of PD-L1 tumor-cell expression (≥25% vs. <25%). DCR-16 was 44.8% (95% CI, 26.5-64.3). Median PFS was 3.5 months (95% CI, 1.9-9.0); median OS was 29.2 months (15.2-not calculable). Twenty-eight (80.0%) patients had treatment-related adverse events [grade 3: 5 (14.3%); no grade 4/5], resulting in discontinuation in 2 (5.7%) patients. HPV-16/18-specific T cells increased on treatment; 4 of 8 evaluable patients had a >2-fold increase in tumor-infiltrating CD8+ T cells. CONCLUSIONS: MEDI0457 plus durvalumab was well tolerated. While the primary efficacy endpoint was not reached, clinical benefit was encouraging.

Clinico-epidemiological profile and management outcome of subungual digital glomus tumor-Indian experience.

BACKGROUND: Glomus tumors are rare tumors arising from the mesenchymal smooth muscle cells of the glomus body. They are extremely painful tumors but because of their subungual location, remain mostly underdiagnosed. AIM: To characterize the demographic, clinical, onychoscopic, radiological features and management outcome of subungual glomus tumor. Material & methods- 15 patients with a total of 16 subungual glomus tumors were evaluated and their demographic data, history, clinical features, investigations, treatment, and follow-up were analyzed. RESULTS: Glomus tumors had a female preponderance (11/15) with thumb being the commonest site. All patients presented with intractable pain. Nail discoloration was observed in 11/16 (68.8%) lesions and nail plate deformity in 6/16 (37.5%) lesions. Common features on onychosocpy were pink glow and linear vascular structures. Doppler sonography and/or magnetic resonance imaging confirmed the diagnosis of glomus tumor in all the lesions. Surgical excision was done under local anesthesia using a trans-ungual approach and no recurrence was seen. CONCLUSIONS: High index of suspicion, meticulous clinical assessment along with radiological investigations can help in the early diagnosis. Complete surgical excision is the treatment of choice to prevent recurrence.

DNA binding, antitubercular, antibacterial and anticancer studies of newly designed piano-stool ruthenium(II) complexes.

The chemotherapeutic potential of ruthenium(II) complexes has recently attracted researchers' interest as antibacterial and anticancer agents. In this study, two novel half-sandwich imine-based Ru complexes ([Ru(p-cymene)Cl(L-1)][PF6] (Ru-1) and [Ru(p-cymene)Cl(L-2)][PF6] (Ru-2)) were reported for their deoxyribonucleic acid (DNA) binding and antitubercular, antibacterial, and anticancer activities. The molecular structure of Ru-2 was obtained by single-crystal X-ray crystallography. DNA interaction studies were conducted by UV-Vis absorbance and fluorescence spectral titration which gave rise to DNA binding constants (Kb) of 1.32 × 106 and 1.82 × 106 for Ru-1 and Ru-2, respectively and the Stern-Volmer binding constant (KSV) values for Ru-1 and Ru-2 were 1.7763 × 104 M-1 and 7.6 × 103 M-1, respectively. The in vitro antitubercular activity was evaluated against Mycobacterium tuberculosis H37Ra. The antibacterial potential of both the Ru-complexes was examined against Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive (Staphylococcus aureus and Bacillus subtilis) bacteria. The half-maximal inhibitory concentration (IC50) values for the antitubercular activity of Ru-1 and Ru-2 were 4.87 ± 1.32 µM and 5.78 ± 0.54 µM, respectively. A cytotoxic study of these complexes was performed against the human breast cancer cell line (MCF-7) and the human embryonic kidney cell line (HEK293) (normal cells). The study revealed meaningful activity of the Ru-1 complex against (cancer) MCF-7 cells, while the viability of HEK293 (normal) cells in the presence of Ru-2 was higher as compared to a reference drug 5FU. We suggest that these kinds of Ru-complexes could have potential for application in metallopharmaceuticals.

Novel gene similar to nitrite reductase (NO forming) plays potentially important role in the latency of tuberculosis.

The development of the latent phenotype of Mycobacterium tuberculosis (Mtb) in the human lungs is the major hurdle to eradicate Tuberculosis. We recently reported that exposure to nitrite (10 mM) for six days under in vitro aerobic conditions completely transforms the bacilli into a viable but non-cultivable phenotype. Herein, we show that nitrite (beyond 5 mM) treated Mtb produces nitric oxide (NO) within the cell in a dose-dependent manner. Our search for the conserved sequence of NO synthesizing enzyme in the bacterial system identified MRA2164 and MRA0854 genes, of which the former was found to be significantly up regulated after nitrite exposure. In addition, the purified recombinant MRA2164 protein shows significant nitrite dependent NO synthesizing activity. The knockdown of the MRA2164 gene at mRNA level expression resulted in a significantly reduced NO level compared to the wild type bacilli with a simultaneous return of its replicative capability. Therefore, this study first time reports that nitrite induces dormancy in Mtb cells through induced expression of the MRA2164 gene and productions of NO as a mechanism for maintaining non-replicative stage in Mtb. This observation could help to control the Tuberculosis disease, especially the latent phenotype of the bacilli.

Iatrogenic Cushing's Syndrome in Patients with Superficial Dermatophytosis.

BACKGROUND: The epidemic-like scenario of superficial fungal infections in India has been complicated by the prescription of systemic and topical potent steroids. As a result, alarming number of patients are presenting with exogenous Cushing's syndrome. METHODS: This cross-sectional study involved 23 patients of superficial dermatophytosis on steroids who presented with clinical features like that of Cushing's syndrome. Their clinical details and laboratory investigations including fungal culture and serum cortisol, were recorded on a pre-designed proforma. RESULTS: There were 23 patients (14 males and 9 females) with mean age of 29.47 ± 15.5 years, majority with extensive tinea cruris and corporis. All of them received oral (Betamethasone) or parenteral corticosteroids along with potent topical steroids (clobetasol propionate and betamethasone valerate) for at least two months. In majority (56.5%), treatment was prescribed by unqualified medical practitioners and in the rest by alternative medical practitioners. Striae, buffalo hump, hirsutism were observed in 16 (69.5%), 15 (65.2%), 13 (56.5%) patients, respectively. Serum cortisol estimation revealed low levels and ranged from 0.66 to 6 µg/ml with a mean of 1.53 ± 1.27 µg/ml (normal 7-25 µg/ml). CONCLUSIONS: Corticosteroids are life saving for many dermatological diseases; their injudicious use (topical, oral, and parenteral) for prolonged periods in the treatment of superficial dermatophytosis can lead to Cushing's syndrome.

Epitope-Specific Antibody Responses to a Plasmodium falciparum Subunit Vaccine Target in a Malaria-Endemic Population.

Circumsporozoite protein (CSP) coats the Plasmodium falciparum sporozoite surface and is a major malaria subunit vaccine target. We measured epitope-specific reactivity to field-derived CSP haplotypes in serum samples from Malian adults and children on a custom peptide microarray. Compared to children, adults showed greater antibody responses and responses to more variants in regions proximal to and within the central repeat region. Children acquired short-lived immunity to an epitope proximal to the central repeat region but not to the central repeat region itself. This approach has the potential to differentiate immunodominant from protective epitope-specific responses when combined with longitudinal infection data.

Tissue specific diversification, virulence and immune response to Mycobacterium bovis BCG in a patient with an IFN-γ R1 deficiency.

Summary: We characterized Mycobacterium bovis BCG isolates found in lung and brain samples from a previously vaccinated patient with IFNγR1 deficiency. The isolates collected displayed distinct genomic and phenotypic features consistent with host adaptation and associated changes in antibiotic susceptibility and virulence traits. Background: We report a case of a patient with partial recessive IFNγR1 deficiency who developed disseminated BCG infection after neonatal vaccination (BCG-vaccine). Distinct M. bovis BCG-vaccine derived clinical strains were recovered from the patient's lungs and brain. Methods: BCG strains were phenotypically (growth, antibiotic susceptibility, lipid) and genetically (whole genome sequencing) characterized. Mycobacteria cell infection models were used to assess apoptosis, necrosis, cytokine release, autophagy, and JAK-STAT signaling. Results: Clinical isolates BCG-brain and BCG-lung showed distinct Rv0667 rpoB mutations conferring high- and low-level rifampin resistance; the latter displayed clofazimine resistance through Rv0678 gene (MarR-like transcriptional regulator) mutations. BCG-brain and BCG-lung showed mutations in fadA2, fadE5, and mymA operon genes, respectively. Lipid profiles revealed reduced levels of PDIM in BCG-brain and BCG-lung and increased TAGs and Mycolic acid components in BCG-lung, compared to parent BCG-vaccine. In vitro infected cells showed that the BCG-lung induced a higher cytokine release, necrosis, and cell-associated bacterial load effect when compared to BCG-brain; conversely, both strains inhibited apoptosis and altered JAK-STAT signaling. Conclusions: During a chronic-disseminated BCG infection, BCG strains can evolve independently at different sites likely due to particular microenvironment features leading to differential antibiotic resistance, virulence traits resulting in dissimilar responses in different host tissues.

Anticancer potential of AgNPs synthesized using Acinetobacter sp. and Curcuma aromatica against HeLa cell lines: A comparative study.

BACKGROUND: Biogenic nanoparticles are gaining attention due to their low toxicity and numerous biomedical applications. Present study aimed to compare the potential anticancer activity of two biogenic silver nanoparticles (bAgNPs and pAgNPs) against human cervical cancer cell lines (HeLa). METHODS: bAgNPs were synthesized using Acinetobacter sp. whereas pAgNPs were synthesized using aqueous root extract of Curcuma aromatica. Effect of these nanoparticles on HeLa cells viability was studied using MTT assay and colony formation assay. Anticancer potential was determined using fluorescence microscopy and flow cytometry studies. Bio-compatibility studies were performed against peripheral blood mononuclear cells (PBMCs). RESULTS: Both the nanoparticles showed 50 % viability of peripheral blood mononuclear cells (PBMCs) when used at high concentration (200 µg/mL). IC50 for bAgNPs and pAgNPs against HeLa cells were 17.4 and 14 µg/mL respectively. Colony formation ability of Hela cells was reduced on treatment with both nanoparticles. Acridine orange and ethidium bromide staining demonstrated that bAgNPs were cytostatic whereas pAgNPs were apoptotic. JC-1 dye staining revealed that the mitochondrial membrane potential was affected on treatment with pAgNPs while it remained unchanged on bAgNPs treatment. Flow cytometry confirmed cell cycle arrest in HeLa cells on treatment with nanoparticles further leading to apoptosis in case of pAgNPs. About 77 and 58 % HeLa cells were found in subG1 phase on treatment with bAgNPs and pAgNPs respectively. bAgNPs showed cytostatic effect on HeLa cells arresting the cell growth in subG1 phase, whereas, pAgNPs triggered death of HeLa cells through mitochondrial membrane potential impairment and apoptosis. CONCLUSION: Overall, bAgNPs and pAgNPs could be safe and showed potential to be used as anticancer nano-antibiotics against human cervical cancer cells.