Clinical and Anatomopathological Evaluation of BALB/c Murine Models Infected with Isolates of Seven Pathogenic Sporothrix Species

Background: Sporotrichosis is a subcutaneous mycosis with worldwide distribution and caused by seven pathogenic species of Sporothrix genus: S. schenckii sensu stricto, S. brasiliensis, S. globosa and S. luriei (clinical clade), and the species S. mexicana, S. pallida and S. chilensis (environmental clade). Isolates of the same species of Sporothrix may have different pathogenicities; however, few isolates of this fungus have been studied. Thus, the aim of this work was to analyze the clinical and anatomopathological changes in immunocompetent and immunosuppressed BALB/c mice infected with clinical and environmental isolates of seven different species of Sporothrix, from both clades. One human clinical isolate of S. schenckii sensu stricto, S. brasiliensis, S. globosa, S. luriei, S. mexicana and S. chilensis species and one environmental isolate of S. pallida were inoculated subcutaneously in immunocompetent mice and the same isolates of S. brasiliensis and S.schenckii sensu stricto were inoculated in immunossupressed mice. Clinical manifestations as external lesions, apathy, and alopecia were observed. At 21, 35, and 49 days after fungal inoculation, four mice from each group were weighed, euthanized and necropsied for evaluation of splenic index, recovery of fungal cells, macroscopic and histopathological analysis of livers, lungs, kidneys, and hearts. The survival assessment was observed for 50 days following inoculation. Our results demonstrated that, clinical S. schenckii isolate, followed by clinical S. mexicana, and environmental S. pallida isolates, the last two, species grouped in the environmental clade, were capable of inducing greater anatomopathological changes in mice, which was reflected in the severity of the clinical signs of these animals. Thus, we reinforce the hypothesis that the pathogenicity of Sporothrix is not only related to the species of this fungus, but also shows variation between different isolates of the same species.

Spleen: an organ of multiple shapes

Introduction: The spleen is located in the upper part of the abdominal cavity. As an organ, the spleen can have various shapes and size. Material and methods: The human spleen was studied in 273 cadavers (154 men and 119 women) who did not have diseases of the spleen. The shape of the spleen was analyzed based on splenic index, Michels classification. Results: The most common shape of the spleen in men is the elongated one. It was encountered in 79 (51.3%) cases out of 154. In women, the most common shape was the intermediate. It was encountered in 51 (42.9%) of the 119 cases. Based on Michels classification the clinoid (wedge) shape was encountered in 102 (37.74%) cases, triangular in 59 (21.83%) and tetrahedral in 30 (11.1%). In 30.26% the shape of the spleen couldn’t be classified according to Michels classification. In 21 cases (7.77%) the spleen had a flat shape; in 27 (9.99%) – dome-shaped; in 1 case (0,37%) – Z-shape; in 18 (6.66%) – round shape; in 6 (2.22%) – irregular shape; in 2 (0.66%) - shape with a node in the hilum; in 1 (0,37%) – rhomboid shape, in 2 (0,74%) – bilobed shape and in 4 cases (1,48%) – lobular shape. The splenic fissures located on the upper edge of the organ were found in 81 (29.91%) cases, and also on the lower edge - in 41 (14.02%) cases. In 13 (4.67%) cases fissures were encountered on both sides. In 148 (51.4%) cases the spleen had no fissures on its surface. Conclusions: The spleen has various shapes beyond the classical wedge, triangular and tetrahedral. All of these shapes do not represent a pathological finding but in certain situation may require further analysis and interpretation depending on the imaging technique and experience of the physician.

Abdominal CT Scan Based Estimation of Splenic Index and Splenic Volume in North Indian Adults

BACKGROUND Exposure of various tropical infections and infestations like tuberculosis, filariasis, malaria, anemia, kala-azar and poor sanitation are major health concerns in Bihar and it’s neighboring state. Spleen size increases in several other diseases e.g., metabolism or storage disorder, malignancies and hematological disorders. Determining the normal parameters of spleen is essential for assessment of splenomegaly in routine ultrasonography (USG) or in the computed tomography (CT) scan. In this study, we wanted to estimate the CT based measurement of splenic dimensions in various planes, splenic index (linear dimension) and splenic volume and also to estimate the relationship of splenic index and splenic volume with age, gender and other splenic dimensions. METHODS It was a retrospective study and data was collected from January 2019 to December 2019. A total of 154 cases (including both genders) of age range 18 - 60 years were collected from the vitrea system (Vitrea software used for the 3-D assessment of splenic volume) and clinical data was collected from the medical record section. CT scan data of only those patients were included who had the clinical history of pain in abdomen, abdominal trauma or other diseases not affecting the spleen. Various measurements including splenic length and thickness at hilum and maximum thickness in axial view and height (maximum interpolar and true vertical height) in coronal views were recorded. RESULTS 154 cases including both gender and age range of 18 - 60 years were included in our study. There were 60 males and 94 females. The average age of the male and female patients was 45.93 +/- 15.19 years and 45.87 +/- 15.12 years respectively. The mean splenic dimensions were 9.03 +/- 1.49 cm in length (axial view), 3.69 +/- 0.05 cm thickness at hilum (axial view) and 9.05 +/- 2.23 cm maximum craniocaudal length and 7.9 +/- 2.96 cm true vertical length (coronal view). Mean splenic index and mean splenic volume were 340.30 +/- 107.39 cm3 and 227.02 +/- 62.22 cm3 respectively. There was statistically significant correlation between splenic volume and maximum craniocaudal length (r2 = 0.4848, P = 0.001), maximum axial length (r2 = 0.4765, P < 0.001) and true vertical length (r2 = 0.3142, P = 0.001) with 95 % confidence interval. For all splenic measurements, there was stronger correlation with maximum craniocaudal length followed by maximum axial length. However, there was no statistically significant correlation either of splenic volume with age in either gender (r2 = 0.019, P = 0.043) or splenic index with age in either gender present (r2 = 0.016, P = 0.059). CONCLUSIONS Maximum splenic length both in coronal (craniocaudal) and axial sections are positively and strongly correlated with splenic volume and splenic index but age of the individuals doesn’t show any correlation either with the splenic volume or with splenic index. Hence, a regional reference of splenic parameters was established with a slightly different range of values reported previously. KEY WORDS Splenic Volume, Splenic Index, Computed Tomography

Tamoxifen Suppresses the Immune Response to Plasmodium berghei ANKA and Exacerbates Symptomatology

Malaria is the most lethal parasitic disease in the world. Mortality and severity in symptoms are higher in men than women, suggesting that oestrogens, which are in higher concentration in females than in males, may regulate the immune response against malaria. Tamoxifen, a selective oestrogen receptor modulator used in breast cancer treatment due to its antagonistic effect on oestrogen receptors α and β, is also studied because of its potential therapeutic use for several parasitic diseases. However, most studies, including one in malaria, have not addressed the immunomodulatory role of tamoxifen. In this work, we evaluated the effect of tamoxifen on the immune response of CBA/Ca mice against Plasmodium berghei ANKA. This study showed for the first time that tamoxifen increased parasite load, aggravated symptoms by decreasing body temperature and body weight, and worsened anaemia. Additionally, tamoxifen significantly increased the splenic index and the percentages of CD4+ and NK+ cells on day eight post-infection. By contrast, tamoxifen decreased both CD8+ and B220+ populations in the spleen and decreased the serum levels of IL-2, IL-6, and IL-17. Our findings support the notion that tamoxifen is a potent immunomodulator in malaria-infected mice and suggest caution when administering it to malaria-infected women with breast cancer.

Insights into irr and rirA gene regulation on the virulence of Brucella melitensis M5-90

Iron is a fundamental element required by most organisms, including Brucella. Several researchers have suggested that the iron response regulator (irr) and rhizobial iron regulator (rirA) genes regulate iron acquisition by Brucella abortus, influencing heme synthesis by and virulence of this pathogen. However, little is known about another Brucella species, Brucella melitensis. In this research, we successfully constructed two mutants: M5-90Δirr and M5-90ΔrirA. The adhesion, invasion, and intracellular survivability of these two mutants were evaluated in RAW264.7 cells infected with 1 × 106 CFU of M5-90Δirr, M5-90ΔrirA, or M5-90. We also tested the sensitivity of cells to hydrogen peroxide and their ability to grow. In addition, the virulence of these two mutants was evaluated in BALB/c mice. The results showed that the ability of these two mutants to invade and adhere inside the murine macrophages RAW264.7 was attenuated but their ability to replicate intracellularly was strengthened, enhancing the resistance to hydrogen peroxide. The M5-90Δirr mutant showed stronger growth ability than the parental strain under iron-limiting conditions. No differences were observed in the number of bacteria in spleen between M5-90 and M5-90Δirr at 7 or 15 days postinfection. However, the number of M5-90ΔrirA in spleen reduced significantly at 15 days postinfection. The splenic index of the M5-90Δirr group is evidently lower than that of M5-90. This is the first report that irr and rirA genes of B. melitensis are associated not only with virulence but also with growth ability. Together, our data suggest that M5-90Δirr is a promising Brucella vaccine candidate.

Iron Restriction Improves Markers of Disease Severity in the Townes Mouse Model of Sickle Cell Anemia

Introduction: Sickle cell anemia (SCA) is caused by mutations in β-globin that result in the production of the abnormal hemoglobin, HbS, with deleterious effects on erythrocyte shape and life span. Because the propensity of erythrocytes to sickle is inversely proportional to the concentration of HbS in the cell, decreasing the mean cellular hemoglobin concentration (MCHC) represents a potential therapeutic approach. Whereas iron restriction in healthy individuals does not alter MCHC, concomitant iron deficiency has been associated with decreased MCHC in SCA patients. Isolated case reports have linked iron restricted erythropoiesis with decreased hemolysis, increased red cell lifespan, and improvement in certain outcomes in SCA patients. We systematically examined the effects of iron restriction on erythropoietic outcomes in SCA utilizing the Townes murine model to investigate the hypothesis that mice with dietary iron deficiency will demonstrate a decreased MCHC, decreased erythrocyte sickling propensity, and improved anemia compared with mice on an iron sufficient diet. Methods: Townes SCA mice were weaned to diets containing either 20 ppm iron (low) or 48 ppm iron (sufficient) and maintained on those diets until sacrifice at 2 months of age. Blood was collected for complete blood count by submandibular or cardiac puncture. Spleen weight was normalized to body weight for calculation of the splenic index. Red cell deformability, defined by the elongation index (EI), and the oxygen pressure at which sickling occurs (point of sickling) during deoxygenation were characterized by oxygenscan ektacytometry using a laser optical rotational red cell analyzer (Mechatronics, The Netherlands). Results: SCA mice fed a 20 ppm low iron diet demonstrate a significant decrease in MCHC compared to SCA mice fed a 48 ppm iron sufficient diet (17.7+1.1 vs 22.7+5.5 g/dL; p <0.05). Mice fed the low iron diet produced more circulating RBCs (5.98+1.03 vs 4.61+0.84 x 106 cells/uL; p <0.05) and a higher hematocrit (39.4+7.1 vs 24.6+4.6%; p <0.05) with a corresponding decrease in splenic index (0.074+0.14 vs 0.087+0.006 g/g body weight; p <0.05) compared to mice fed the iron sufficient diet. RBCs from mice fed the low iron diet showed a significant decrease in the oxygen pressure at the point of sickling compared to RBCs from mice fed the iron sufficient diet (31.9+4.7 vs 40.6+4.6 mmHg; p <0.01). The low iron diet also resulted in an overall improvement in deformability as evidenced by an increase in the EI minimum compared to the iron sufficient diet (0.25+.07 vs 0.12+.03 au; p <0.01). No significant differences between groups were found in maximum deformability (EI max). The improved EI minimum translates to a decreased change in elongation (Delta EI), defined as EI max minus EI min, in RBCs from mice fed the low iron diet compared to mice fed the iron sufficient diet (0.11+.08 vs .23+.02 au; p <0.01). Conclusions: Decreased dietary iron intake is associated with a decrease in the MCHC in Townes SCA mice. The lower MCHC is accompanied by an amelioration of anemia, decreased extramedullary erythropoiesis, improved RBC deformability and a shift in the initiation of sickling to lower oxygen pressures during deoxygenation. We speculate that iron restriction may decrease the clinical complications arising from both hemolysis and sickling in SCA. Future studies will utilize this model system to further characterize the effects of manipulating iron homeostasis on erythropoietic outcomes in SCA. Disclosures Fleming: Protagonist: Membership on an entity's Board of Directors or advisory committees; Silence Therapeutics: Consultancy; Ultragenyx: Consultancy.

Hematologic changes and splenic index on malaria mice models given Syzygium cumini extract as an adjuvant therapy

Aim: This research aimed to determine the efficacy of Syzygium cumini L. as an adjuvant therapy on blood changes and splenic index of mice model malaria. Materials and Methods: Mice were infected intraperitoneally with 0.2 ml red blood cell (RBC) that contains 1×106 Plasmodium berghei. 35 mice were divided into seven treatment groups: Group K0: Mice were not infected; K1: Mice were infected; K2: Mice were infected and given chloroquine; P1: Mice were infected and given S. cumini leaf extract; P2: Mice were infected and given chloroquine and also S. cumini leaf extract; P3: Mice was infected and given S. cumini stem bark extract; and P4: Mice were infected and given chloroquine and S. cumini stem bark extract. Treatment was given for 4 days 24 h post-P. berghei infection. 21st day post-P. berghei infection, blood was taken from the heart for hematological examination, and the spleen was taken to examine the splenic index and also to measure the weight and length of the spleen. Hematological data and splenic index were analyzed by analysis of variance test, and if there is a difference, the test is continued by Duncan's multiple range test with 5% level. Results: The K0 group has normal hemoglobin (HGB), RBC, and hematocrit (HCT) and significantly different (p<0.05) than other groups. HGB, RBC, and HCT of K1 group were under normal range, lowest, and significantly different (p<0.05) than other groups. Mean corpuscular volume and mean corpuscular HGB values of K2 groups showed a decrease. The number of leukocytes, lymphocytes, and monocytes of K1 groups was increasing and significantly different (p<0.05) with K2 and treatment group. The length, width, weight, and splenic index of K1 group were significantly different (p<0.05) with K0 group. K2 and treatment groups showed that the length and width of spleens were significantly different (p<0.05) with K1. Conclusion: The combination of chloroquine with leaf and chloroquine with stem bark extract of S. cumini as adjuvant therapy may increase the amount of erythrocyte; decrease the number of leukocytes, lymphocytes, and monocytes; and decrease the length, width, and splenic index on malaria mice models.

CT SCAN BASED EVALUATION OF SPLENIC INDEX IN PATIENTS FROM LUCKNOW AND ADJOINING AREAS

Introduction: The Indian subcontinent is an endemic zone for diseases like malaria and filaria which alter the size of spleen to a variable extent. In order to identify and examine the spleen accurately, the modes of investigation that are at our disposal are Ultrasonography, Computerised Tomography, Magnetic Resonance Imaging and Radionuclide Scan. Linear dimensions are of limited use as a guide to normal splenic size on CT because the spleen is irregular in shape and is obliquely oriented within the left upper quadrant. A more accurate approach to the assessment of splenic size is the splenic index. Sufficient and reliable data for its normal values are lacking. The objective of the present study was to generate normative data of splenic index which could be used as a research and clinical tool. Material & Methods: It was an open randomized study carried out on 80 adults between 20 to 70 years of age. Spleen was identified in each transverse, coronal and sagittal section image of helical CT scan. Results: The average splenic index, calculated as a product of the three measured linear dimensions of spleen i.e. length, breadth and thickness, in males was 369.89 cm³ and in females 298.61 cm³. The mean splenic index was calculated as 342.62 cm³. Conclusion: The results can be used for evaluating patients with suspected splenomegaly, in certain clinical situations where objective measurement of splenic dimensions and comparison with normal values is required, and in drafting of government policies and programs related to malaria and filaria control where these normal values of splenic index help by identifying populations more susceptible to such infections.