Loxosceles venom-induced cytokine activation, hemolysis, and acute kidney injury

Herein, we describe a confirmed case of Loxosceles spider bite that illustrates the critical complications seen in loxoscelism, including skin necrosis, rhabdomyolysis, hemolysis, coagulopathy, acute kidney failure, and electrolyte disorders. Upon initial assessment, laboratory studies revealed the following: the white blood cell count was 29 400 WBCs/mm(3), hemoglobin was 9.2g/dL, and the platelet count was 218000cells/mm(3). Coagulation studies revealed the following: international normalized ratio, 1.83; activated partial-thromboplastin time, 62s; D-dimer, 600 ng/mL (normal range < 500 ng/mL); free protein S, 37% (normal range = 64-114%); protein C, negative; and antithrombin III, negative. Various serum levels were abnormal: urea, 110mg/dL; creatinine, 3.1 mg/dL; indirect bilirubin, 3.8 mg/dL; creatine kinase, 1631 U/L, lactate dehydrogenase, 6591 U/L; potassium 6.2mmol/L. Urine tests were positive for hemoglobin and bilirubin. In addition, concentrations of interleukin-6 and tumor necrosis factor-alpha were notably elevated in the serum. In conclusion, physicians must be alert to the possibility of loxoscelism when a patient presents with the clinical and laboratory findings described above, especially if the patient resides in an endemic area. Advances in our understanding of multiple pathways and mediators that orchestrate the response to Loxosceles venom might reveal new possibilities for the management of loxoscelism. (C) 2007 Elsevier Ltd. All rights reserved.

Melatonin reduces the severity of experimental amoebiasis

Background: Melatonin has immunomodulatory effects but very little is known about its influence in protozoan infections, such as Entamoeba histolytica, which causes amoebiasis, a disease with significant morbidity and mortality. In this study, we evaluated the effects of exogenous melatonin interference in experimental amoebiasis and on interactions between human blood cells and E. histolytica trophozoites. Methods: The effect of melatonin was investigated in models of experimental amoebiasis in hamsters and rats by evaluating the area of necrosis induced by E. histolytica. The activity of melatonin on the interactions between leukocytes and amoebae was determined by examining leukophagocytosis. For in vitro tests, polymorphonuclear and mononuclear human blood leucocytes were incubated with E. histolytica trophozoites. Results: The areas of amoebic necrosis were significantly reduced in animals treated with melatonin. Melatonin treatment increased leukophagocytosis but was associated with a greater number of dead amoebae. Conclusions: These results suggest that melatonin may play a beneficial role in the control of amoebic lesions, raising the possibility that this drug may be used as an adjuvant in anti-amoebic therapy.

Urogenital tuberculosis in a patient with AIDS: an unusual presentation

Background A 38-year-old man with AIDS presented to hospital with a 3-month history of fevers, bilateral lumbar pain, dysuria and increased urinary frequency. Six years earlier he had received 6 months` treatment for pulmonary tuberculosis. At presentation, he was on antiretroviral therapy with a combination of efavirenz, stavudine and lamivudine. Investigations Physical examination, evaluation of HIV viral load, CD4 count, measurement of serum hemoglobin concentration, white blood cell count, urinalysis, urine culture for usual pathogens, direct smear and urine culture for Mycobacterium tuberculosis, chest radiography, abdominal CT, measurement of serum creatinine concentration and estimated creatinine clearance. Diagnosis Urogenital tuberculosis. Management The patient`s symptoms and radiological abnormalities persisted despite antibiotic therapy for presumed bacterial infection. After urine culture had confirmed M. tuberculosis infection, he was administered pharmacological treatment comprising isoniazid, rifampin, pyrazinamide and ethambutol for 2 months, with isoniazid and rifampin given for a further 7 months. His symptoms improved within a few days of initiating treatment. Six months after treatment started, CT revealed a nonfunctioning right kidney and a functional left kidney with areas of scarring. The patient refused right nephrectomy, and completed his pharmacological treatment. No evidence of disease recurrence was observed during 2 years of follow-up.

Outcome of acute myeloid leukemia patients with hyperleukocytosis in Brazil

Acute myeloid leukemia (AML) with a high white blood cell (WBC) count at presentation has been associated with an increased early mortality rate, usually secondary to leukostasis. However, the value of the WBC count at which there is a high risk of early death (ED) and the efficiency of supportive treatments remain unclear. In this report, a series of 187 consecutive adult patients with AML in our institution was reviewed. The outcome of 40 patients with WBC above 50 x 10(9) L(-1) (hyperleukocytosis) was compared to 147 patients with a leukocyte count lower than 50 x 10(9) L(-1). The group with hyperleukocytosis showed a significantly shorter OS (P < 0.0001) and a higher rate of ED (P = 0.0008). Even when the data from ED patients were removed from analysis, we still detected a shorter OS in patients with hyperleukocytosis (P = 0.0049), which suggests that high WBC number influences long-term survival, and not only ED. We also observed higher lactic dehydrogenase (LDH) and serum creatinine levels in the group of patients with hyperleukocytosis (P = 0.0003 and 0.0406, respectively). Besides considering all the patients with ED, we could observe higher levels of lactic dehydrogenase, a serum creatinine and nitrogen urea (P = 0.0056, P = 0.0008 and P < 0.0001, respectively). Pulmonary involvement was more frequent in patients with ED (P = 0.0277). In conclusion, hyperleukocytosis confers a poorer prognosis in patients with AML.

Tetrasomy 8 in a patient with chronic lymphocytic leukemia

We report a case of a 47-year-old man diagnosed with chronic lymphocytic leukemia (CLL) with two extra copies of chromosome 8. Classical cytogenetic analysis by the immunostimulatory combination of DSP30 and interleukin 2 showed tetrasomy of chromosome 8 in 60% of the metaphase cells (48,XY,+8,+8[12]/46,XY[8]). Spectral karyotype analysis confirmed the abnormality previously seen by G banding. Additionally, interphase fluorescence in situ hybridization using an LSI CEP 8 probe performed on peripheral blood cells without any stimulant agent showed tetrasomy of chromosome 8 in 54% of analyzed cells (108 of 200). To our knowledge, tetrasomy 8 as the sole chromosomal abnormality in CLL has not been previously described. The prognostic significance of tetrasomy 8 in CLL remains to be elucidated. However, the patient has been followed up in the outpatient hospital since 2004 without any therapeutic intervention and has so far remained stable. (C) 2010 Elsevier Inc. All rights reserved.

Low mRNA Expression of the Apoptosis-Related Genes CASP3, CASP8, and FAS Is Associated With Low Induction Treatment Response in Childhood Acute Lymphoblastic Leukemia (ALL)

Background. Defects in apoptosis signaling have been considered to be responsible for treatment failure in many types of cancer, although with controversial results. The objective of the present study was to assess the expression profile of key apoptosis-related genes in terms of clinical and biological variables and of the survival of children with acute lymphoblastic leukemia (ALL). Procedure. The levels of mRNA expression of the apoptosis-related genes CASP3, CASP8, CASP9, FAS, and BCL2 were analyzed by quantitative real-time PCR in consecutive samples from 139 consecutive children with ALL at diagnosis treated by the Brazilian protocol (GBTLI-ALL 99). Gene expression levels and clinical and biological features were compared by the Mann-Whitney test. Event-free survival (EFS) was calculated by Kaplan-Meier plots and log-rank test. Results. A significant correlation was detected between CASP3, CASP8, CASP9, and FAS expression levels (P<0.01) in ALL samples. Higher levels of BCL2 were significantly associated with white blood cell (WBC) count <50,000/mm(3) at diagnosis (P=0.01) and low risk group classification (P=0.008). Lower expression levels of CASP3, CASP8 and FAS gene were associated with a poor response at day 7 according the GBTLI-ALL 99 protocol (P=0.03, P=0.02 and P=0.008, respectively). There was a relationship between FAS gene expression lower than the 75th percentile and lower 5-year EFS (P=0.02). Conclusion. These findings suggest an association between lower expression levels of the pro-apoptotic genes and a poor response to induction therapy at day 7 and prognosis in childhood ALL. Pediatr Blood Cancer 2010;55:100-107. (C) 2010 Wiley-Liss, Inc.

Evaluation of the Expression and Protective Potential of Leptospiral Sphingomyelinases

Leptospirosis is a zoonotic disease of global distribution, which affects both animals and humans. Pathogenic leptospires, the bacteria that cause this disease, require iron for their growth, and these spirochetes probably use their hemolysins, such as the sphingomyelinases, as a way to obtain this important nutrient from host red blood cells during infection. We expressed and purified the leptospiral sphingomyelinases Sph1, Sph2, Sph4, and SphH in a heterologous system. However, the recombinant proteins were not able to lyse sheep erythrocytes, despite having regular secondary structures. Transcripts for all sphingomyelinases tested were detected by RT-PCR analyses, but only Sph2 and SphH native proteins could be detected in Western blot assays using Leptospira whole extracts as well as in renal tubules of infected hamsters. Moreover, antibodies present in the serum of a human patient with laboratory-confirmed leptospirosis recognized Sph2, indicating that this sphingomyelinase is expressed and exposed to the immune system during infection in humans. However, in an animal challenge model, none of the sphingomyelinases tested conferred protection against leptospirosis.

Ectonucleotidase activities are altered in serum and platelets of L-NAME-treated rats

It is well known that hypertension is closely associated to the development of vascular diseases and that the inhibition of nitric oxide biosynthesis by administration of N omega-Nitro-L-arginine methyl ester hydrochloride (L-NAME) leads to arterial hypertension. In the vascular system, extracellular purines mediate several effects: thus, ADP is the most important platelet agonist and recruiting agent, while adenosine, all end product Of nucleotide metabolism, is a vasodilator and inhibitor of platelet activation and recruitment. Members of several families of enzymes, known as ectonucleotidases, including E-NTPDases (ecto-nucleoside triphosphate diphosphohydrolase), E-NPP (ecto-nucleotide pyrophosphatase/phosphodiesterase) and 5`-nucleotidase are able to hydrolyze extracellular nucleotides until their respective nucleosides. We investigated the ectonuclectidase activities of serum and platelets from rats made hypertensive by oral administration of L-NAME (30 mg/kg/day for 14 days or 30 mg/kg/day for 14 days Plus 7 days of L-NAME washout, in the drinking water) in comparison to normotensive control rats. L-NAME promoted a significant rise in systolic blood pressure from 112 +/- 9.8 to 158 +/- 23 mmHg. The left ventricle weight index (LVWI) was increased in rats treated with L-NAME for 14 days when compared to control animals. In Serum samples, ATP, ADP and AMP hydrolysis were reduced by about 27%, 36% and 27%, respectively. In platelets, the decrease in ATP, ADP and AMP hydrolysis Was approximately 27%, 24% and 32%, respectively. All parameters recovered after 7 days of L-NAME washout. HPLC demonstrated a reduction in ADP, AMP and hypoxanthine levels by about 64%, 69% and 87%, respectively. In this study, we showed that ectonucleotidase activities are decreased in serum and platelets from L-NAME-treated rats, which should represent an additional risk for the development of hypertension. The modulation of ectonucleotidase activities may represent an approach to antihypertensive therapy via inhibition of spontaneous platelet activation and recruitment, as well as thrombus formation. (C) 2008 Elsevier Inc. All rights reserved.

The role of hemocytes in the immunity of the spider Acanthoscurria gomesiana

Invertebrates protect themselves against microbial infection through cellular and humoral immune defenses. Since the available information on the immune system of spiders is scarce, the main goat of the present study was to investigate the role of hemocytes and antimicrobial peptides (AMPs) in defense against microbes of spider Acanthoscurria gomesiana. We previously described the purification and characterization of two AMPs from the hemocytes of naive spider A. gomesiana, gomesin and acanthoscurrin. Here we show that 57% of the hemocytes store both gomesin and acanthoscurrin, either in the same or in different granules. Progomesin labeling in hemocyte granules indicates that gomesin is addressed to those organelles as a propeptide. In vivo and in vitro experiments showed that lipopolysaccharide (LPS) and yeast caused the hemocytes to migrate. Once they have reached the infection site, hemocytes may secrete coagulation cascade components and AMPs to cell-free hemolymph. Furthermore, our results suggest that phagocytosis is not the major defense mechanism activated after microbial challenge. Therefore, the main reactions involved in the spider immune defense might be coagulation and AMP secretion. (C) 2007 Elsevier Ltd. All rights reserved.

Effects of cyclooxygenase inhibitors on parasite burden, anemia and oxidative stress in murine Trypanosoma cruzi infection

Prostaglandins are known to be produced by macrophages when challenged with Trypanosoma cruzi, the etiological agent of Chagas` disease. It is not known whether these lipid mediators play a role in oxidative stress in host defenses against this important protozoan parasite. In this study, we demonstrated that inducible cyclooxygenase-mediated prostaglandin production is a key chemical mediator in the control of parasite burden and erythrocyte oxidative stress during T. cruzi infection in C57BL/6 and BALB/c mice, prototype hosts for the study of resistance and susceptibility in murine Chagas` disease. The results suggested the existence of at least two mechanisms of oxidative stress, dependent or independent with regard to the nitric oxide and cyclooxygenase pathway, where one or the other is more evident depending on the mouse strain.

Hematologically important mutations: X-linked chronic granulomatous disease (third update)

Chronic granulomatous disease (CGD) is an immunodeficiency disorder affecting about 1 in 250,000 individuals. The disease is caused by a lack of superoxide production by the leukocyte enzyme NADPH oxidase. Superoxide is used to kill phagocytosed micro-organisms in neutrophils, eosinophils, monocytes and macrophages. The leukocyte NADPH oxidase is composed of five subunits, of which the enzymatic component is gp91-phox, also called Nox2. This protein is encoded by the CYBB gene on the X chromosome. Mutations in this gene are found in about 70% of all CGD patients. This article lists all mutations identified in CYBB in the X-linked form of CGD. Moreover, apparently benign polymorphisms in CYBB are also given, which should facilitate the recognition of future disease-causing mutations. (C) 2010 Elsevier Inc. All rights reserved.

Leukotrienes Produced in Allergic Lung Inflammation Activate Alveolar Macrophages

It has been well-documented that leukotrienes (LTs) are released in allergic lung inflammation and that they participate in the physiopathology of asthma. A role for LTs in innate immunity has recently emerged: Cys-LTs were shown to enhance Fc gamma R-mediated phagocytosis by alveolar macrophages (AMs). Thus, using a rat model of asthma, we evaluated Fc gamma R-mediated phagocytosis and killing of Klebsiella pneumoniae by AMs. The effect of treatment with a cys-LT antagonist (montelukast) on macrophage function was also investigated. Male Wistar rats were immunized twice with OVA/alumen intraperitoneally and challenged with OVA aerosol. After 24 h, the animals were killed, and the AMs were obtained by bronchoalveolar lavage. Macrophages were cultured with IgG-opsonized red blood cells (50: 1) or IgG-opsonized K. pneumoniae (30: 1), and phagocytosis or killing was evaluated. Leukotriene C(4) and nitric oxide were quantified by the EIA and Griess methods, respectively. The results showed that AMs from sensitized and challenged rats presented a markedly increased phagocytic capacity via Fc gamma R (10X compared to controls) and enhanced killing of K. pneumoniae (4X higher than controls). The increased phagocytosis was inhibited 15X and killing 3X by treatment of the rats with montelukast, as compared to the non-treated group. cys-LT addition increased phagocytosis in control AMs but had no effect on macrophages from allergic lungs. Montelukast reduced nitric oxide (39%) and LTC(4) (73%). These results suggest that LTs produced during allergic lung inflammation potentiate the capacity of AMs to phagocytose and kill K. pneumonia via Fc gamma R. Copyright (C) 2010 S. Karger AG, Basel

5-Lipoxygenase plays a role in the control of parasite burden and contributes to oxidative damage of erythrocytes in murine Chagas` disease

Chagas` disease is accompanied by severe anemia and oxidative stress, which may contribute to mortality. In this study, we investigated the role of 5-lipoxygenase (5-LO) in the control of parasitism and anemia associated with oxidative damage of erythrocytes in experimental Trypanosoma cruzi infection. Wild-type C57BL/6, 129Sv mice treated or not with nordihydroguaiaretic acid (NDGA, 5-LO inhibitor), mice lacking the 5-LO enzyme gene (5-LO(-/-)) and inducible nitric oxide synthase gene (iNOS(-/-)) were infected with the Y strain of T cruzi. impairment of 5-LO resulted in increased numbers of trypomastigote forms in the blood and amastigote forms in the heart of infected mice. We assessed oxidative stress in erythrocytes by measuring oxygen uptake, induction time and chemiluminescence following treatment with tert-butyl hydroperoxide (TBH). Our results show that 5-LO metabolites increased lipid peroxidation levels in erythrocytes during the early phase of murine T cruzi infection. NDGA treatment reduced oxidative damage of erythrocytes in C57BL/6 T cruzi-infected mice but not in C57BL/6 iNOS-/- infected mice, showing that the action of NDGA is dependent on endogenous nitric oxide (NO). In addition, our results show that 5-LO metabolites do not participate directly in the development of anemia in infected mice. We conclude that 5-LO products may not only play a major role in controlling heart tissue parasitism, i.e., host resistance to acute infection with T cruzi in vivo, but in the event of an infection also play an important part in erythrocyte oxidative stress, an NO-dependent effect. (C) 2009 Elsevier B.V. All rights reserved.

Boosting systemic and secreted antibody responses in mice orally immunized with recombinant Bacillus subtilis strains following parenteral priming with a DNA vaccine encoding the enterotoxigenic Escherichia coli (ETEC) CFA/I fimbriae B subunit

Recombinant Bacillus subtilis strains, either spores or vegetative cells, may be employed as safe and low cost orally delivered live vaccine vehicles. In this study, we report the use of an orally delivered B. subtilis vaccine strain to boost systemic and secreted antibody responses in mice i.m. primed with a DNA vaccine encoding the structural subunit (CfaB) of the CFA/I fimbriae encoded by enterotoxigenic Escherichia coli (ETEC), an important etiological agent of diarrhea among travelers and children living in endemic regions. DBA/2 female mice submitted to the prime-boost immunization regimen developed synergic serum (IgG) and mucosal (IgA) antibody responses to the target CfaB antigen. Moreover, in contrast to mice immunized only with one vaccine formulation, sera harvested from prime-boosted vaccinated individuals inhibited adhesion of ETEC cells to human red blood cells. Additionally, vaccinated dams conferred full passive protection to suckling newborn mice challenged with a virulent ETEC strain. Taken together the present results further demonstrate the potential use of recombinant B. subtilis strains as an alternative live vaccine vehicle. (C) 2008 Elsevier Ltd. All rights reserved.

Impaired phagocytosis by alveolar macrophages from diabetic rats is related to the deficient coupling of LTs to the Fe gamma R signaling cascade

Diabetic individuals are more susceptible to infections and this seems to be related to impaired phagocyte function. Alveolar macrophages (AMs) are the first barrier to prevent respiratory infections Leukotrienes (LTs) increase AM phagocytic activity via Fc gamma R. In this study, we compared AMs from diabetic and nondiabetic rats for phagocytosis via Fc gamma R and the roles of LTs and insulin Diabetes was induced in male Wistar rats by alloxan (42 mg/kg, i.v); macrophages were obtained by bronchoalveolar lavage and IgG-opsonised sheep red blood cells (IgG-SRBC) were used as targets. LTs were added to the AMs 5 min before the addition of IgG-SRBC. AMs were treated with a LT synthesis inhibitor (zileuton, 10 mu M), or antagonists of the LTB(4) receptor (CP105 696, 10 mu M) cys-LT receptor (MK571, 10 mu M), 30 or 20 min before the addition of IgG-SRBC, respectively. We found that the phagocytosis of IgG-SRBC by AMs from diabetic rats is impaired compared with non-diabetic rats. Treatment with the LT inhibitor/antagonists significantly reduced AM phagocytosis in non-diabetic but not diabetic rats. During the phagocytosis of IgG-SRBC LTB(4) and LTC(4) were produced by AMs from both groups. The addition of exogenous LTB(4) or LTD(4) potentiated phagocytosis similarly in both groups Phagocytosis was followed by the phosphorylation of PKC-delta. ERK and Akt This was reduced by zileuton treatment in AMs from non-diabetic but not diabetic rats The addition of insulin to AMs further increased the phagocytosis by increasing PKC-delta phosphorylation These results suggest that the impaired phagocytosis found in AMs from diabetic rats is related to a deficient coupling of LTs to the Fc gamma R signaling cascade and that insulin has a key role in this coupling An essential role for insulin in Innate immunity is suggested (C) 2010 Elsevier Ltd. All rights reserved.