Cleverton Roberto de Andrade

Evaluation of Antimalarial Activity and Toxicity of a New Primaquine Prodrug

Plasmodium vivax is the most prevalent of the five species causing malaria in humans. The current available treatment for P. vivax malaria is limited and unsatisfactory due to at least two drawbacks: the undesirable side effects of primaquine (PQ) and drug resistance to chloroquine. Phenylalanine-alanine-PQ (Phe-Ala-PQ) is a PQ prodrug with a more favorable pharmacokinetic profile compared to PQ. The toxicity of this prodrug was evaluated in in vitro assays using a human hepatoma cell line (HepG2), a monkey kidney cell line (BGM), and human red blood cells deficient in the enzyme glucose-6-phosphate-dehydrogenase (G6PD). In addition, in vivo toxicity assays were performed with rats that received multiple doses of Phe-Ala-PQ to evaluate biochemical, hematological, and histopathological parameters. The activity was assessed by the inhibition of the sporogonic cycle using a chicken malaria parasite. Phe-Ala-PQ blocked malaria transmission in Aedes mosquitoes. When compared with PQ, it was less cytotoxic to BGM and HepG2 cells and caused less hemolysis of G6PD-deficient red blood cells at similar concentrations. The prodrug caused less alteration in the biochemical parameters than did PQ. Histopathological analysis of the liver and kidney did show differences between the control and Phe-Ala-PQ-treated groups, but they were not statistically significant. Taken together, the results highlight the prodrug as a novel lead compound candidate for the treatment of P. vivax malaria and as a blocker of malaria transmission.

Experimental development of bisphosphonate-related osteonecrosis of the jaws in rodents

Osteonecrosis of the jaw (ONJ) following the use of bisphosphonates has become of increased interest in the scientific community, due in particular to its as‐yet‐unsolved pathogenesis. An experimental model of ONJ was induced in normal male rats [alendronate (ALN); 1 mg/Kg/day; n = 10] and matched controls (saline solution; n = 10). After 60 days of drug treatment, all animals were subjected to extractions of the left first lower molars and were euthanized at 3 and 28 days postsurgery. The following analyses were performed: (i) descriptive and quantitative (scores) histological evaluation, (ii) stereometry of distal sockets and (iii) biochemical measurement of C‐telopeptide cross‐linked collagen type I (CTX) and bone‐specific alkaline phosphatase (BALP). The results showed that 28 days postsurgery the animals treated with ALN had areas of exposed and necrotic bone, associated with significant infection, especially in the interalveolar septum area and crestal regions, compared with controls. The levels of CTX, BALP and bone volume, as well as the degrees of inflammation and vascularization, were significantly reduced in these animals. Therefore, analysis of the data presented suggests that ALN therapy is associated with the development of osteonecrosis in the jaws of rodents after tooth extraction.

The NLRP3 inflammasome contributes to host protection during Sporothrix schenckii infection

Sporotrichosis is a mycosis caused by fungi from the Sporothrix schenckii species complex, whose prototypical member is Sporothrix schenckii sensu stricto. Pattern recognition receptors (PRRs) recognize and respond to pathogen‐associated molecular patterns (PAMPs) and shape the following adaptive immune response. A family of PRRs most frequently associated with fungal recognition is the nucleotide‐binding oligomerization domain‐like receptor (NLR). After PAMP recognition, NLR family pyrin domain‐containing 3 (NLRP3) binds to apoptosis‐associated speck‐like protein containing a caspase recruitment domain (ASC) and caspase‐1 to form the NLRP3 inflammasome. When activated, this complex promotes the maturation of the pro‐inflammatory cytokines interleukin‐1β (IL‐1β) and IL‐18 and cell death through pyroptosis. In this study, we aimed to evaluate the importance of the NLRP3 inflammasome in the outcome of S. schenckii infection using the following three different knockout (KO) mice: NLRP3−/−, ASC−/− and caspase‐1−/−. All KO mice were more susceptible to infection than the wild‐type, suggesting that NLRP3‐triggered responses contribute to host protection during S. schenckii infection. Furthermore, the NLRP3 inflammasome appeared to be critical for the ex vivo release of IL‐1β, IL‐18 and IL‐17 but not interferon‐γ. Additionally, a role for the inflammasome in shaping the adaptive immune response was suggested by the lower frequencies of type 17 helper T (Th17) cells and Th1/Th17 but not Th1 cells in S. schenckii‐infected KO mice. Overall, our results indicate that the NLRP3 inflammasome links the innate recognition of S. schenckii to the adaptive immune response, so contributing to protection against this infection.

Comparative efficacy and toxicity of two vaccine candidates against Sporothrix schenckii using either Montanide™ Pet Gel A or aluminum hydroxide adjuvants in mice

Sporotrichosis is an important zoonosis in Brazil and the most frequent subcutaneous mycosis in Latin America, caused by different Sporothrix species. Currently, there is no effective vaccine available to prevent this disease. In this study, the efficacy and toxicity of the adjuvant Montanide™ Pet Gel A (PGA) formulated with S. schenckii cell wall proteins (ssCWP) was evaluated and compared with that of aluminum hydroxide (AH). Balb/c mice received two subcutaneous doses (1st and 14th days) of either the unadjuvanted or adjuvanted vaccine candidates. On the 21st day, anti-ssCWP antibody levels (ELISA), the phagocytic index, as well as the ex vivo release of IFN-γ, IL-4, and IL-17 by splenocytes and IL-12 by peritoneal macrophages were assessed. Cytotoxicity of the vaccine formulations was evaluated in vitro and by histopathological analysis of the inoculation site. Both adjuvanted vaccine formulations increased anti-ssCWP IgG, IgG1, IgG2a, and IgG3 levels, although IgG2a levels were higher in response to PGA+CWP100, probably contributing to the increase in S. schenckii yeast phagocytosis by macrophages in the opsonophagocytosis assay when using serum from PGA+CWP100-immunized mice. Immunization with AH+CWP100 led to a mixed Th1/Th2/Th17 ex vivo cytokine release profile, while PGA+CWP100 stimulated a preferential Th1/Th2 profile. Moreover, PGA+CWP100 was less cytotoxic in vitro, caused less local toxicity and led to a similar reduction in fungal load in the liver and spleen of S. schenckii- or S. brasiliensis-challenged mice as compared with AH+CWP100. These results suggest that PGA may be an effective and safe adjuvant for a future sporotrichosis vaccine.

Oral Verruciform Xanthoma: A Case Report and Literature Review

Oral verruciform xanthoma represents an uncommon entity, which affects mainly oral mucosa. This paper presents the major clinical and histological features of oral verruciform xanthoma and reports a case on the tongue. The differential diagnosis and a literature review are also provided in light of recent information.

Experimental osteonecrosis: development of a model in rodents administered alendronate.

The main objective of this study was to cause bisphosphonate-related osteonecrosis of the jaws to develop in a rodent model. Adult male Holtzman rats were assigned to one of two experimental groups to receive alendronate (AL; 1 mg/kg/week; n = 6) or saline solution (CTL; n = 6). After 60 days of drug therapy, all animals were subjected to first lower molar extraction, and 28 days later, animals were euthanized. All rats treated with alendronate developed osteonecrosis, presenting as ulcers and necrotic bone, associated with a significant infection process, especially at the inter-alveolar septum area and crestal regions. The degree of vascularization, the levels of C-telopeptide cross-linked collagen type I and bone-specific alkaline phosphatase, as well as the bone volume were significantly reduced in these animals. Furthermore, on radiographic analysis, animals treated with alendronate presented evident sclerosis of the lamina dura of the lower first molar alveolar socket associated with decreased radiographic density in this area. These findings indicate that the protocol developed in the present study opens new perspectives and could be a good starting model for future property design.

Effects of Chronic Stress and Alendronate Therapy on the Osseointegration of Titanium Implants

PURPOSES The purposes of this study were to evaluate the influence of chronic stress (CS) on implant osseointegration and also to analyze whether alendronate (ALN) therapy could prevent these eventual stress-negative effects. MATERIALS AND METHODS Adult male Holtzmann rats were assigned to one of the four experimental groups: AL (ALN; 1 mg/kg/week; n = 12), ALS (ALN + CS; 1 mg/kg/week; n = 12), CTL (sterile physiological saline; n = 12), or CTLS (sterile physiological saline + CS; n = 12). After 58 days of drug therapy, the ALS and CTLS groups were exposed to CS, and 2 days later all animals underwent tibial implant installation. The animals were euthanized 28 days following the operative surgical procedure. RESULTS It was observed that the CTLS group presented an impairment of bone metabolism represented by lowest levels of bone-specific alkaline phosphatase and bone area fraction occupancy values. Furthermore, these animals presented a higher proportion of empty osteocytic lacunae. In contrast, the ALN therapy showed increased osseointegration and torque value parameters, regardless of stress exposition. CONCLUSIONS Analysis of the data presented suggests that CS partially impairs the osseointegration of tibial implants and that ALN therapy is able to prevent these negative effects.

Hydroxymethylnitrofurazone treatment in indeterminate form of chronic Chagas disease: Reduced intensity of tissue parasitism and inflammation-A histopathological study

Hydroxymethylnitrofurazone (NFOH) is a nitrofurazone prodrug effective in vivo during acute infections, and it has less hepatotoxicity effect than the standard drug benznidazole (BZN) which has been used during short‐ and long‐term treatment. In the present study, we induced the indeterminate form of Chagas disease in mice with a Y strain of Trypanosoma cruzi and analysed the histopathological data about the effects of NFOH and BZN on different tissues, including the heart, skeletal muscle, liver, kidney, colon, spleen and brain. After infection, BALB/c mice were treated with NFOH (150 mg/kg) and BZN (60 mg/kg) for 60 days and then submitted to immunosuppression using dexamethasone (5 mg/kg) for 14 days. Two trained analysts, as part of a blind evaluation, examined the results using serial sections of 3 mm diameter in two different moments. The results showed reactivation of the disease only in the infected nontreated group (POS). After treatment, amastigote nests were found in the heart, colon, liver and skeletal muscle in the POS group and in the heart and liver of the BZN group. Interestingly, amastigote nests were not found in the NFOH and NEG groups. The histopathological analysis showed fewer tissue lesions and parasite infiltrates in the NFOH group when compared with the BZN and POS groups. We have not observed any increase in the levels of hepatocellular injury biomarkers (AST/ALT) in the NFOH group. These in vivo studies show the potential for NFOH as an effective and safe compound useful as an anti‐T. cruzi agent.

In vivo antileishmanial activity and histopathological evaluation in Leishmania infantum infected hamsters after treatment with a furoxan derivative

N-oxide derivatives compounds such as furoxan and benzofuroxan are promising scaffolds for designing of new antileishmanial drugs. A series of furoxan (1,2,5-oxadiazole 2-N-oxide) (compounds 4a-b, and 14a-f) and benzofuroxan (benzo[c][1,2,5]oxadiazole1-N-oxide) (compounds 8a-c) derivatives were evaluated against in vitro cultured L. infantum promastigotes and amastigotes. The compounds exhibited activity against promastigote and intracellular amastigote forms with EC50 values ranging from 2.9 to 71.2μM and 2.1 to 18.2μM, respectively. The most promising compound, 14e, showed good antileishmanial activity (EC50=3.1μM) against intracellular amastigote forms of L. infantum with a selectivity index, based on murine macrophages (SI=66.4), almost 3-times superior to that presented by the standard drug amphotericin B (AmpB). The efficacy of 14e to eliminate the parasites in vivo was also demonstrated. Treatment of L. infantum-infected hamsters with compound 14e at 3.0mg/Kg/day led to a meaningful reduction of parasite load in spleen (49.9%) and liver (54.2%), respectively; these data were corroborated by histopathological analysis, which also revealed reduction in the number of inflammatory cells in the liver of the treated animals. Moreover, histological analysis of the spleen and kidney of treated animals did not reveal alterations suggestive of toxic effects. The parasite load reduction might be related to NO production, since this molecule is a NO-donor. We observed neither side effects nor elevation of hepatic/renal biomarker levels in the plasma. The data herein presented suggest that the compound should be considered in the development of new drugs for treatment of visceral leishmaniasis.

Pathological Mandibular Fracture Associated With Diffuse Large B-Cell Lymphoma in HIV-Positive Patient

This article describes the occurrence of diffuse large B-cell lymphoma in a 39-year-old human immunodeficiency virus-positive patient. The patient sought medical care complaining of increased volume in the right mandibular angle and imaging tests showed an extensive radiolucency with undefined boundaries compromising the mandibular border. After the incisional biopsy, the patient had a pathological fracture in the region, which was properly treated in a second surgical procedure using a 2.4-mm reconstruction plate. Immunohistochemical analysis revealed positive marking for CD3, CD79a, Ki67, and Epstein-Barr virus-encoded RNA. The treatment consisted of concurrent antiretroviral therapy with chemotherapy with rituximab combined with cyclophosphamide, doxorubicin, vincristine, and prednisone. Examinations of images (2 years postoperatively) revealed complete bone repair and absence of injury recurrence. This work is important because it describes an unusual location of diffuse large B-cell lymphoma and shows the importance of diagnosis and treatment of the injury at an early stage in order to promote the prognosis and survival of patients.