Evaluation of Acute and Sub-Acute Toxicity, Oxidative Stress and Molecular Docking of Two Nitrofuranyl Amides as Promising Anti-Tuberculosis Agents.

Tuberculosis (TB) remains a widespread infectious disease and one of the top 10 causes of death worldwide. Nevertheless, despite significant advances in the development of new drugs against tuberculosis, many therapies and preventive measures do not lead to the expected favorable health results for various reasons. The aim of this study was to evaluate the acute and sub-acute toxicity and oxidative stress of two selected nitrofuranyl amides with high in vitro antimycobacterial activity. In addition, molecular docking studies were performed on both compounds to elucidate the possibilities for further development of new anti-tuberculosis candidates with improved efficacy, selectivity, and pharmacological parameters. Acute toxicity tests showed that no changes were observed in the skin, coat, eyes, mucous membranes, secretions, and vegetative activity in mice. The histological findings include features consistent with normal histological architecture without being associated with concomitant pathological conditions. The observed oxidative stress markers indicated that the studied compounds disturbed the oxidative balance in the mouse liver. Based on the molecular docking, compound DO-190 showed preferable binding energies compared to DO-209 in three out of four targets, while both compounds showed promising protein-ligand interactions. Thus, both studied compounds displayed promising activity with low toxicity and can be considered for further evaluation and/or lead optimization.

Effect of Chitosan-Diosgenin Combination on Wound Healing.

The difficult-to-heal wounds continue to be a problem for modern medicine. Chitosan and diosgenin possess anti-inflammatory and antioxidant effects making them relevant substances for wound treatment. That is why this work aimed to study the effect of the combined application of chitosan and diosgenin on a mouse skin wound model. For the purpose, wounds (6 mm diameter) were made on mice's backs and were treated for 9 days with one of the following: 50% ethanol (control), polyethylene glycol (PEG) in 50% ethanol, chitosan and PEG in 50% ethanol (Chs), diosgenin and PEG in 50% ethanol (Dg) and chitosan, diosgenin and PEG in 50% ethanol (ChsDg). Before the first treatment and on the 3rd, 6th and 9th days, the wounds were photographed and their area was determined. On the 9th day, animals were euthanized and wounds' tissues were excised for histological analysis. In addition, the lipid peroxidation (LPO), protein oxidation (POx) and total glutathione (tGSH) levels were measured. The results showed that ChsDg had the most pronounced overall effect on wound area reduction, followed by Chs and PEG. Moreover, the application of ChsDg maintained high levels of tGSH in wound tissues, compared to other substances. It was shown that all tested substances, except ethanol, reduced POx comparable to intact skin levels. Therefore, the combined application of chitosan and diosgenin is a very promising and effective medication for wound healing.

Photodynamic Inactivation of Bovine Coronavirus with the Photosensitizer Toluidine Blue O.

Coronaviruses (CoVs) belong to the group of enveloped positive-sense single-strand RNA viruses and are causative agents of respiratory, gastro-intestinal, and central nervous systems diseases in many host species, i.e., birds, mammals, and humans. Beta-CoVs revealed a great potential to cross the barrier between species by causing three epidemics/pandemics among humans in the 21st century. Considering the urgent need for powerful antiviral agents for decontamination, prevention, and treatment of BCoV infections, we turned our attention to the possibility of photodynamic inactivation with photosensitizers in combination with light irradiation. In the present study, we evaluated, for the first time, the antiviral activity of toluidine blue O (TBO) against Beta-coronavirus 1 (BCoV) in comparison to methylene blue (MB). First, we determined the in vitro cytotoxicity of MB and TBO on the Madin-Darby bovine kidney (MDBK) cell line with ISO10993-5/Annex C. Thereafter, BCoV was propagated in MDBK cells, and the virus titer was measured with digital droplet PCR, TCID50 assay and plaque assay. The antiviral activity of non-toxic concentrations of TBO was estimated using the direct inactivation approach. All effects were calculated in MAPLE 15® mathematical software by developing programs for non-linear modeling and response surface analysis. The median inhibitory concentration (IC50) of TBO after 72 h of incubation in MDBK cells was 0.85 µM. The antiviral activity of TBO after the direct inactivation of BCoV (MOI = 1) was significantly stronger than that of MB. The median effective concentration (EC50) of TBO was 0.005 µM. The cytopathic effect decreased in a concentration-dependent manner, from 0.0025 to 0.01 µM, and disappeared fully at concentrations between 0.02 and 0.3 µM of TBO. The number of virus particles also decreased, depending on the concentration applied, as proven by ddPCR analysis. In conclusion, TBO exhibits significant potential for direct inactivation of BCoV in vitro, with a very high selectivity index, and should be subjected to further investigation, aiming at its application in veterinary and/or human medical practice.

Micellar Curcumin Substantially Increases the Antineoplastic Activity of the Alkylphosphocholine Erufosine against TWIST1 Positive Cutaneous T Cell Lymphoma Cell Lines.

Cutaneous T-cell lymphoma (CTCL) is a rare form of cancer with local as well as systemic manifestations. Concomitant bacterial infections increase morbidity and mortality rates due to impaired skin barrier and immune deficiency. In the current study, we demonstrated that the in vitro anti-lymphoma potential of erufosine is diminished by TWIST1 expression and micellar curcumin substantially increases its antineoplastic activity. Pharmacokinetic analysis showed that the micellar curcumin (MCRM) used in our study was characterized by low zeta potential, slow release of curcumin, and fast cell membrane penetration. The combination ratio 1:4 [erufosine:MCRM] achieved strong synergism by inhibiting cell proliferation and clonogenicity. The combined antiproliferative effects were calculated using the symbolic mathematical software MAPLE 15. The synergistic combination strongly decreased the expression of TWIST1 and protein kinase B/Akt as proven by western blotting. Significant reductions in NF-κB activation, induction of apoptosis, and altered glutathione levels were demonstrated by corresponding assays. In addition, the synergistic combination enhanced the anti-staphylococcal activity and prevented biofilm formation, as shown by crystal violet staining. Taken together, the above results show that the development of nanotechnological treatment modalities for CTCL, based on rational drug combinations exhibiting parallel antineoplastic and antibacterial effects, may prove efficacious.

In Vivo Toxicity, Redox-Modulating Capacity and Intestinal Permeability of Novel Aroylhydrazone Derivatives as Anti-Tuberculosis Agents.

The emergence and spread of Mycobacterium tuberculosis strains resistant to many or all anti-tuberculosis (TB) drugs require the development of new compounds both efficient and with minimal side effects. Structure-activity-toxicity relationships of such novel, structurally diverse compounds must be thoroughly elucidated before further development. Here, we present the aroylhydrazone compounds (3a and 3b) regarding their: (i) acute and subacute toxicity in mice; (ii) redox-modulating in vivo and in vitro capacity; (iii) pathomorphology in the liver, kidney, and small intestine tissue specimens; and (iv) intestinal permeability. The acute toxicity test showed that the two investigated compounds exhibited low toxicity by oral and intraperitoneal administration. Changes in behavior, food amount, and water intake were not observed during 14 days of the oral administration at two doses of 1/10 and 1/20 of the LD50. The histological examination of the different tissue specimens did not show toxic changes. The in vitro antioxidant assays confirmed the ex vivo results. High gastrointestinal tract permeability at all tested pH values were demonstrated for both compounds. To conclude, both compounds 3a and 3b are highly permeable with low toxicity and can be considered for further evaluation and/or lead optimization.

In Vitro Antineoplastic and Antiviral Activity and In Vivo Toxicity of Geum urbanum L. Extracts.

This study evaluated the in vitro antineoplastic and antiviral potential and in vivo toxicity of twelve extracts with different polarity obtained from the herbaceous perennial plant Geum urbanum L. (Rosaceae). In vitro cytotoxicity was determined by ISO 10993-5/2009 on bladder cancer, (T-24 and BC-3C), liver carcinoma (HEP-G2) and normal embryonic kidney (HEK-293) cell lines. The antineoplastic activity was elucidated through assays of cell clonogenicity, apoptosis induction, nuclear factor kappa B p65 (NFκB p65) activation and total glutathione levels. Neutral red uptake study was applied for antiviral activity. The most promising G. urbanum extract was analyzed by UHPLC-HRMS. The acute in vivo toxicity analysis was carried out following OEDC 423. The ethyl acetate extract of aerial parts (EtOAc-AP) exhibited the strongest antineoplastic activity on bladder cancer cell lines (IC50 = 21.33-25.28 µg/mL) by inducing apoptosis and inhibiting NFκB p65 and cell clonogenicity. EtOAc and n-butanol extracts showed moderate antiviral activity against human adenovirus type 5 and human simplex virus type I. Seventy four secondary metabolites (gallic and ellagic acid derivatives, phenolic acids, flavonoids, etc.) were identified in EtOAc-AP by UHPLC-HRMS. This extract induced no signs of acute toxicity in liver and kidney specimens of H-albino mice in doses up to 210 mg/kg. In conclusion, our study contributes substantially to the detailed pharmacological characterization of G. urbanum, thus helping the development of health-promoting phytopreparations.

Subungual Exostosis in a Young Soccer Player.

BACKGROUND: Subungual exostosis is a relatively uncommon, benign osteocartilaginous tumor of the distal phalanx of the toes or fingers in young adults, considered as a rare variant of osteochondroma. Differential diagnoses include subungual verruca (viral wart), pyogenic granuloma, osteochondroma, amelanotic subungual melanoma and glomus tumour. Misdiagnosis and total onychodystrophy frequently occur as a result of late treatment or inadequate treatment strategy. Dermoscopy could be a useful technique, involved in the diagnostic process, although X-ray examination and histopathology are mandatory for the diagnosis. CASE REPORT: We report a rare case of subungual exostosis of the great toe associated with repeated trauma of the nail bed. The lack of radiographic and histopathological examination could lead to misdiagnosis and inadequate treatment. Although completely benign, subungual exostosis should be considered in differential diagnosis of nail bed tumors in young adults, in order to avoid associated complications and unneeded aggressive surgical interventions. CONCLUSION: Complete excision of the lesion and delicate separation from the underlying nail bed structures results in total resolve of the problem, by providing the lowest risk of recurrences.

A Patient with Multiple Keratinocytic Cancers (MKC): Uncommon Presentation in a Bulgarian Patient.

Keratinocyte skin cancers, including basal cell carcinoma (BCC) and squamous cell carcinoma (SCC), are the most common cancer occurring in people with fair skin, worldwide. Despite all known triggers, several suggested contributors are still investigated. We will focus our attention on the personal history of previous cancers and radiation exposure as occupational risk factors, as in the presented case. We report a patient, with multiple BCCs, and subsequent occurrence of a SCC on photo-exposed area of the face, as we want to emphasize the importance of strict following up of these patients, regarding the risk for developing new tumors in short periods of time, no matter if the triggering exposure factor is known from the history, or not. Although keratinocytes tumours are associated with the low mortality rate, we focus the attention on the fact, that the history of non-melanoma skin cancer is associated with increased mortality.

Subungual Squamous Cell Carcinoma Associated with Long Standing Onychomycosis: Aggressive Surgical Approach with a Favourable Outcome.

BACKGROUND: Tumours of the nail bed are rare. Squamous cell carcinoma is the most frequent among them. Chronic infection, chemical or physical trauma/microtrauma, genetic disorders such as congenital ectodermal dysplasia, radiation, tar, arsenic or exposure to minerals, sun exposure, immunosuppression, and previous HPV infection have all been discussed as etiologic factors. The diagnosis is often delayed because of the variety of clinical manifestations, often resembling benign or common infectious processes. Rapidly growing ulcerative lesions should also be considered as potential malignancy. Furthermore, a lack of antifungal or antibacterial treatment response is the most indicative symptom, always requiring subungual biopsy. Early diagnosis is of great importance for therapeutic effectiveness. CASE PRESENTATION: We present a case of subungual squamous cell carcinoma, associated with long-lasting onychomycosis in a 76-year-old female patient, treated with amputation of the distal phalanx and the distal part of the proximal phalanx. CONCLUSION: Although there are no available data in the literature to confirm or reject the contribution of the chronic nail infection to the malignant process, we emphasise the importance of this co-existence regarding the possible disguising of the malignant process. An early biopsy of a chronic persistent nail lesion may be preventive and beneficial regarding avoiding more aggressive treatments and achieving a favourable prognosis.

Antioxidant response and biocompatibility of curcumin-loaded triblock copolymeric micelles.

To evaluate the safety profile of cationic micelles, based on triblock copolymer poly(dimethylaminoethyl methacrylate)-poly(e-caprolactone)-poly(dimethylaminoethyl methacrylate) (PDMAEMA9- PCL70-PDMAEMA9), the effects of empty (PM) and curcumin loaded micelles (PM-Curc) on nonenzyme induced lipid peroxidation (LPO) in vitro, hemolytic activity and morphological changes in some organs after repeated intraperitoneal administration in vivo were studied. To induce LPO, rat liver microsomes were incubated with a solution of iron sulfate and ascorbinic acid (Fe2+/AA). The effect of empty PM (40 and 100 µg/ml), PM-Curc and free curcumin (both at 3.48 and 8.7 µg curcumin/ml) was assessed at 20 min incubation time. In the non-enzyme induced LPO model, the investigated substances at all concentrations significantly decreased the formation of malondialdehyde (MDA), compared to the Fe2+/AA induced LPO group. According to the results it can be concluded that curcumin alone and loaded in PM, exert significant antioxidant activity. In the biocompatibility safety studies, the mean hemolytic index for polymeric carrier was less than 2%, indicating it was non-hemolytic. The general appearance of the organ tissues from Wistar rats, treated in vivo with curcumin loaded PM was similar to that of controls, thus showing no apparent toxicity after repeated 14-days treatment.

In vitro and in vivo toxicity evaluation of cationic PDMAEMA-PCL-PDMAEMA micelles as a carrier of curcumin.

Polymeric micelles have attracted significant attention because of their potential application as promising drug-delivery systems. In the present study cationic micelles, based on triblock copolymer poly(dimethylaminoethyl methacrylate) - poly(e-caprolactone) - poly(dimethylaminoethyl methacrylate) were prepared and loaded with curcumin. In vitro cytotoxicity of empty and curcumin loaded polymer micelles was investigated on two cell culture models, human hepatoma cell line HEP G2 and freshly isolated rat hepatocytes, following their viability and lactate dehydrogenase (LDH) leakage. MTT dye reduction assay and LDH release study showed that empty cationic micelles did not cause significant changes in cell viability and membrane integrity at the concentration range from 10.0 to 80.0 µg/ml. Our special attention was focused on the effects of empty and curcumin loaded micelles on oxidative stress markers malondialdehyde (MDA) and reduced glutathione (GSH). The increase in the micelles concentration to 100 µg/ml was accompanied by GSH depletion and increased levels of MDA production in isolated rat hepatocytes. The in vivo toxicity of polymeric micelles was examined in male Wistar rats. The results showed that neither single (7.5 mg/kg, i.p.), nor repeated (3.5 mg/kg, i.p., 14 days) exposure to empty or curcumin loaded polymeric micelles induced any toxicity changes, e.g. hematopoietic and liver tissue damages.

Relapsing advanced metatypical basal cell carcinomas (MTBCC) of the face: surgical modalities.

Metatypical basal cell carcinoma (MTBCC) represents a high-risk type of cutaneous tumour. We report about three different patients with relapsing advanced large MTBCC: one of the scalp and two of the cheek region. Such patients required in most of the cases a complex surgical approach to achieve a stable and complete remission. In the first presented patient a combination of flaps and grafts has been performed. We describe tailored surgical approaches. By this contrivance it is possible to treat even elderly patients with exposed bone after complete excision effectively and safe. Interdisciplinary team work is for the benefit of these patients.

Multiple primary cutaneous melanomas in patients with FAMMM syndrome and sporadic atypical mole syndrome (AMS): what's worse?

Atypical Mole Syndrome is the most important phenotypic risk factor for cutaneous melanoma, a malignancy that accounts for about 80% of deaths from skin cancer. Since early diagnosis of melanoma is of great prognostic relevance, the identification of Atypical Mole Syndrome carriers (sporadic and familial) is essential, as well as the recommendation of preventative measures that must be undertaken by these patients.We report two rare cases concerning patients with multiple primary skin melanomas in the setting of a familial and a sporadic syndrome of dysplastic nevi: the first patient is a 67-year-old patient with a history of multiple superficial spreading melanomas localized on his back. The second patient presented with multiple primary melanomas in advanced stage in the context of the so-called sporadic form of the syndrome of dysplastic nevi-AMS (atypical mole syndrome). In the first case, excision of the melanomas was carried out with an uneventful post-operative period. In the second case, disseminated metastases were detected, involving the right fibula, the abdominal cavity as well as multiple lesions in the brain. The patient declined BRAF mutation tests as well as chemotherapy or targeted therapies, and suffered a rapid deterioration in his general condition leading to death. We classified the second case as a sporadic form of the atypical mole syndrome, associated with one nodular and two superficial spreading melanomas.There are no data in the literature to allow us to understand if, in patients with multiple primary melanomas, there is any difference in terms of prognosis between those with and without a family history of a similar phenotype. To answer this and other questions related to these rare cases, further studies with a significant number of patients should be carried out.

Anorectal malignant melanoma in a hemorrhoidal nodule: a diagnostic and therapeutic problem.

Anorectal malignant melanoma (ARMM) is an extremely rare condition, often misdiagnosed and mistreated until development of metastatic disease. Clinical presentation mimicking hemorrhoids is a well-known pitfall. We present a male patient with hemorrhoidal nodules who was referred to the policlinic of dermatology for management of anal pruritus. A dark macule was detected over one of the hemorrhoidal nodules histologically verified as melanoma. Subsequent CT and PET/CT showed lymph nodes involvement and the patient underwent wide local excision (WSE) followed by abdominoperineal resection (APR). The rarity of ARMM does not allow for establishment of a validated staging system, placebo-controlled treatment trials and management guidelines adoption. The current treatment for the condition is surgical excision, using different techniques according to the stage of the disease and depth of invasion. The prognosis and overall survival are poor, but recent genetic studies give promising results for molecular targeting. Awareness for this disease is indispensable, as early recognition could result in improved survival and quality of life.

Nerve growth factor immunoreactivity of mast cells in acute involuted human thymus.

The acute involution of the thymus is induced by either exogenous or endogenous factors, including some infections (infection type involution). The present study was focused on both detection and immunocytochemical analysis of NGF immunopositive mast cells in child thymus with acute infection-induced involution. Autopsy thymus specimens from children with infection diseases (Sepsis, Encephalomyelitis, Varicella) were examined at light and electron microscopic level and compared to normal infantile thymuses. We observed a redistribution of NGF immunopositive mast cells in infection-affected child thymus, which lobular architecture was collapsed. A positive correlation between the degree of the involutive changes, increased distribution and enhanced NGF immunoreactivity of mast cells was defined. The possible involvement of NGF immunopositive mast cells in the process of acute thymus involution is discussed.

Altered levels of nerve growth factor in the thymus of subjects with myasthenia gravis.

We have previously reported that nerve growth factor (NGF), a polypeptide known for its neurotrophic activities, is also involved in the differentiation and survival of immune cells, and that NGF and its high-affinity receptor are present in the thymus. We here demonstrate that the thymus of humans affected by myasthenia gravis (MG) contains significant concentrations of NGF. These observations support our hypothesis of a role for NGF in the thymus and suggest that the changes observed in the thymus of subject with MG may have functional significance.