Neutrophil-derived microparticles induce myeloperoxidase-mediated damage of vascular endothelial cells.

BACKGROUND: Upon activation neutrophil releases microparticles - small plasma membrane vesicles that contain cell surface proteins and cytoplasmic matter, with biological activities. In this study we investigated the potential role of myeloperoxidase in the endothelial cell injury caused by neutrophil-derived microparticles. RESULTS: Microparticles were produced by activating human neutrophils with a calcium ionophore and characterized by flow cytometry and transmission and scanning electron microscopy. Myeloperoxidase activity was measured by luminol-dependent chemiluminescence. Neutrophil microparticles-induced injuries and morphological alterations in human umbilical vein endothelial cells (HUVECs) were evaluated by microscopy and flow cytometry. Neutrophil microparticles were characterized as structures bounded by lipid bilayers and were less than 1 µm in diameter. The microparticles also expressed CD66b, CD62L and myeloperoxidase, which are all commonly expressed on the surface of neutrophils, as well as exposition of phosphatidylserine. The activity of the myeloperoxidase present on the microparticles was confirmed by hypochlorous acid detection. This compound is only catalyzed by myeloperoxidase in the presence of hydrogen peroxide and chloride ion. The addition of sodium azide or taurine inhibited and reduced enzymatic activity, respectively. Exposure of HUVEC to neutrophil microparticles induced a loss of cell membrane integrity and morphological changes. The addition of sodium azide or myeloperoxidase-specific inhibitor-I consistently reduced the injury to the endothelial cells. Taurine addition reduced HUVEC morphological changes. CONCLUSIONS: We have demonstrated the presence of active myeloperoxidase in neutrophil microparticles and that the microparticle-associated myeloperoxidase cause injury to endothelial cells. Hence, the microparticle-associated myeloperoxidase-hydrogen peroxide-chloride system may contribute to widespread endothelial cell damage in conditions of neutrophil activation as observed in vasculitis and sepsis.

The spleen is the graveyard of CD4+ cells in patients with immunological failure of visceral leishmaniasis and AIDS.

BACKGROUND: Visceral leishmaniasis (VL), or kala-azar, is a common comorbidity in patients with AIDS in endemic areas. Many patients continue to experiences relapses of VL despite virological control, but with immunological failure. These patients remain chronically symptomatic with hypersplenism, for example with anemia, leukopenia, and thrombocytopenia, and are at risk of severe co-infection due to low CD4+ count. Therefore, in this study, splenectomized patients with VL and HIV infection were investigated to understand why the CD4+ count fails to recover in these patients, evaluating the importance of spleen mass for hypersplenism and immunological failure. METHODS: From a retrospective open cohort of 13 patients who had previously undergone splenectomy as salvage therapy for relapsing VL, 11 patients with HIV infection were investigated. This study compared the patients' complete blood cell count (CBC) and CD4+ and CD8+ cell counts before and after splenectomy with respect to spleen weight. RESULTS: CBC was substantially improved after splenectomy, indicating hypersplenism. However, to the best of our knowledge, this is the first study to show that spleen mass is strongly and negatively correlated with CD4+ cell count (ρ = -0.71, P = 0.015). CONCLUSIONS: This finding was unexpected, as the spleen is the most extensive lymphoid tissue and T-lymphocyte source. After reviewing the literature and reasoning, we hypothesized that the immunological failure was secondary to CD4+ loss initially by apoptosis in the spleen induced by productive HIV infection and, subsequently, by pyroptosis sustained by parasitic infection in spleen macrophages.

Pharmacokinetics, Dose-Proportionality, and Tolerability of Intravenous Tanespimycin (17-AAG) in Single and Multiple Doses in Dogs: A Potential Novel Treatment for Canine Visceral Leishmaniasis.

In the New World, dogs are considered the main reservoir of visceral leishmaniasis (VL). Due to inefficacies in existing treatments and the lack of an efficient vaccine, dog culling is one of the main strategies used to control disease, making the development of new therapeutic interventions mandatory. We previously showed that Tanespimycin (17-AAG), a Hsp90 inhibitor, demonstrated potential for use in leishmaniasis treatment. The present study aimed to test the safety of 17-AAG in dogs by evaluating plasma pharmacokinetics, dose-proportionality, and the tolerability of 17-AAG in response to a dose-escalation protocol and multiple administrations at a single dose in healthy dogs. Two protocols were used: Study A: four dogs received variable intravenous (IV) doses (50, 100, 150, 200, or 250 mg/m2) of 17-AAG or a placebo (n = 4/dose level), using a cross-over design with a 7-day "wash-out" period; Study B: nine dogs received three IV doses of 150 mg/m2 of 17-AAG administered at 48 h intervals. 17-AAG concentrations were determined by a validated high-performance liquid chromatographic (HPLC) method: linearity (R2 = 0.9964), intra-day precision with a coefficient of variation (CV) ≤ 8%, inter-day precision (CV ≤ 20%), and detection and quantification limits of 12.5 and 25 ng/mL, respectively. In Study A, 17-AAG was generally well tolerated. However, increased levels of liver enzymes-alanine aminotransferase (ALT), aspartate aminotransferase (AST), and gamma-glutamyl transferase (GGT)-and bloody diarrhea were observed in all four dogs receiving the highest dosage of 250 mg/m2. After single doses of 17-AAG (50-250 mg/m2), maximum plasma concentrations (Cmax) ranged between 1405 ± 686 and 9439 ± 991 ng/mL, and the area under the curve (AUC) plotting plasma concentration against time ranged between 1483 ± 694 and 11,902 ± 1962 AUC 0-8 h µg/mL × h, respectively. Cmax and AUC parameters were dose-proportionate between the 50 and 200 mg/m2 doses. Regarding Study B, 17-AAG was found to be well tolerated at multiple doses of 150 mg/m2. Increased levels of liver enzymes-ALT (28.57 ± 4.29 to 173.33 ± 49.56 U/L), AST (27.85 ± 3.80 to 248.20 ± 85.80 U/L), and GGT (1.60 ± 0.06 to 12.70 ± 0.50 U/L)-and bloody diarrhea were observed in only 3/9 of these dogs. After the administration of multiple doses, Cmax and AUC 0-48 h were 5254 ± 2784 µg/mL and 6850 ± 469 µg/mL × h in plasma and 736 ± 294 µg/mL and 7382 ± 1357 µg/mL × h in tissue transudate, respectively. In conclusion, our results demonstrate the potential of 17-AAG in the treatment of CVL, using a regimen of three doses at 150 mg/m2, since it presents the maintenance of high concentrations in subcutaneous interstitial fluid, low toxicity, and reversible hepatotoxicity.

Inflammation in disseminated lesions: an analysis of CD4+, CD20+, CD68+, CD31+ and vW+ cells in non-ulcerated lesions of disseminated leishmaniasis.

Disseminated leishmaniasis (DL) differs from other clinical forms of the disease due to the presence of many non-ulcerated lesions (papules and nodules) in non-contiguous areas of the body. We describe the histopathology of DL non-ulcerated lesions and the presence of CD4-, CD20-, CD68-, CD31- and von Willebrand factor (vW)-positive cells in the inflamed area. We analysed eighteen biopsies from non-ulcerated lesions and quantified the inflamed areas and the expression of CD4, CD20, CD68, CD31 and vW using Image-Pro software (Media Cybernetics). Diffuse lymphoplasmacytic perivascular infiltrates were found in dermal skin. Inflammation was observed in 3-73% of the total biopsy area and showed a significant linear correlation with the number of vW+ vessels. The most common cells were CD68+ macrophages, CD20+ B-cells and CD4+ T-cells. A significant linear correlation between CD4+ and CD20+ cells and the size of the inflamed area was also found. Our findings show chronic inflammation in all DL non-ulcerated lesions predominantly formed by macrophages, plasmacytes and T and B-cells. As the inflamed area expanded, the number of granulomas and extent of the vascular framework increased. Thus, we demonstrate that vessels may have an important role in the clinical evolution of DL lesions.

Transplantation of bone marrow mononuclear cells reduces mortality and improves renal function on mercury-induced kidney injury in mice.

OBJECTIVE: Recent studies have demonstrated the therapeutic effects of bone marrow-derived cells in tissue regeneration. The aim of this study was to investigate the effects of bone marrow mononuclear cell (BMMC) transplantation in a mouse model of acute renal failure (ARF) induced by mercuric chloride. METHODS: BMMC was isolated from male BALB/c mice and injected into female mice treated with a lethal dose (LD90) of mercuric chloride. Survival rate, histopathological analysis, and assessment of urea, creatinine, sodium, potassium, and mercury levels were carried out. RESULTS: Cellular therapy with BMMC significantly reduced the mortality induced by mercuric chloride (p < 0.05). This finding correlated with a decrease in serum levels of urea (p = 0.04) and potassium (p < 0.01). However, no differences in renal morphology were observed when BMMC-treated and control group were compared. CONCLUSION: Transplanted BMMC improve renal function and reduce mortality and, therefore, may represent a new therapeutic alternative to treat ARF.

Acute tubular necrosis and renal failure in patients with glomerular disease.

Renal failure is common in patients with glomerular disease. Although renal failure may result from the glomerular lesion itself, it is also observed in patients with minimal glomerular alterations. Degenerative changes and necrosis of the tubular epithelium are common findings in kidney biopsies from these patients. The aim of this work is to examine the association between acute tubular necrosis (ATN) and renal failure in patients with glomerulopathy and to estimate the relationship between the degree of ATN and renal failure in these patients. Data on age, sex, presence of nephrotic syndrome, and renal failure were recorded for 149 patients, who underwent a renal biopsy for the diagnosis of glomerulopathy. The biopsies were reviewed, and ATN, when present, was classified as one of four grades depending on its intensity. The mean age of the patients was 21 ± 16 years. Eighty patients (54%) were male, 43 (42%) had renal failure, 104 (72%) had nephrotic syndrome, and 66 (45%) had minimal change disease or focal segmental glomerulosclerosis. ATN was present in 115 (77%) patients. The frequency of renal failure was directly correlated with the intensity of ATN [odds ratio (OR) of 26.0 for patients with grade 2 lesions and OR of 45.5 for patients with grade 3 lesions]. ATN is a common finding in the biopsies of patients with glomerulopathy. The severity of ATN is directly associated with the frequency of renal failure in these patients.

Schistosomal glomerulopathy and changes in the distribution of histological patterns of glomerular diseases in Bahia, Brazil.

Distinct patterns of glomerular lesions, including membranoproliferative glomerulonephritis and focal segmental glomerulosclerosis, are associated with infection by Schistosoma mansoni or Schistosoma japonicum. Evidence suggests that immune complex deposition is the main mechanism underlying the different forms of schistosomal glomerulonephritis and that immune complex deposition may be intensified by portal hypertension. The relationship between focal segmental glomerulosclerosis and schistosomiasis remains poorly understood. A clinicopathologic classification of schistosomal glomerulopathies was proposed in 1992 by the African Association of Nephrology. In Brazil, mass treatment with oral medications has led to a decrease in the occurrence of schistosomal glomerulopathy. In a survey of renal biopsies performed in Salvador, Brazil, from 2003-2009, only 24 (4%) patients were identified as positive for S. mansoni infection. Among these patients, only one had the hepatosplenic form of the disease. Focal segmental glomerulosclerosis was found in seven patients and membranoproliferative glomerulonephritis was found in four patients. Although retrospective studies on the prevalence of renal diseases based on kidney biopsies may be influenced by many patient selection biases, a change in the distribution of glomerulopathies associated with nephrotic syndrome was observed along with a decline in the occurrence of severe forms of schistosomiasis.

An experimental protocol for the establishment of dogs with long-term cellular immune reactions to Leishmania antigens.

Domestic dogs are considered to be the main reservoirs of zoonotic visceral leishmaniasis. In this work, we evaluated a protocol to induce Leishmania infantum/Leishmania chagasi-specific cellular and humoral immune responses in dogs, which consisted of two injections of Leishmania promastigote lysate followed by a subcutaneous inoculation of viable promastigotes. The primary objective was to establish a canine experimental model to provide positive controls for testing immune responses to Leishmania in laboratory conditions. After inoculation of viable promastigotes, specific proliferative responses of peripheral blood mononuclear cells (PBMCs) to either Leishmania lysate or recombinant proteins, the in vitro production of interferon-γ by antigen-stimulated PBMCs and a significant increase in circulating levels of anti-Leishmania antibodies were observed. The immunized dogs also displayed positive delayed-type hypersensitivity reactions to Leishmania crude antigens and to purified recombinant proteins. An important finding that supports the suitability of the dogs as positive controls is that they remained healthy for the entire observation period, i.e., more than seven years after infection. Following the Leishmania antigen lysate injections, the infection of dogs by the subcutaneous route appears to induce a sustained cellular immune response, leading to an asymptomatic infection. This provides a useful model for both the selection of immunogenic Leishmania antigens and for immunobiological studies on their possible immunoprotective activities.

Oral Tolerance Induced by Heat Shock Protein 65-Producing Lactococcus lactis Mitigates Inflammation in Leishmania braziliensis Infection.

Cutaneous leishmaniasis caused by L. braziliensis induces a pronounced Th1 inflammatory response characterized by IFN-γ production. Even in the absence of parasites, lesions result from a severe inflammatory response in which inflammatory cytokines play an important role. Different approaches have been used to evaluate the therapeutic potential of orally administrated heat shock proteins (Hsp). These proteins are evolutionarily preserved from bacteria to humans, highly expressed under inflammatory conditions and described as immunodominant antigens. Tolerance induced by the oral administration of Hsp65 is capable of suppressing inflammation and inducing differentiation in regulatory cells, and has been successfully demonstrated in several experimental models of autoimmune and inflammatory diseases. We initially administered recombinant Lactococcus lactis (L. lactis) prior to infection as a proof of concept, in order to verify its immunomodulatory potential in the inflammatory response arising from L. braziliensis. Using this experimental approach, we demonstrated that the oral administration of a recombinant L. lactis strain, which produces and secretes Hsp65 from Mycobacterium leprae directly into the gut, mitigated the effects of inflammation caused by L. braziliensis infection in association or not with PAM 3CSK4 (N-α-Palmitoyl-S-[2,3-bis(palmitoyloxy)-(2RS)-propyl]-L-cysteine, a TLR2 agonist). This was evidenced by the production of anti-inflammatory cytokines and the expansion of regulatory T cells in the draining lymph nodes of BALB/c mice. Our in vitro experimental results suggest that IL-10, TLR-2 and LAP are important immunomodulators in L. braziliensis infection. In addition, recombinant L. lactis administered 4 weeks after infection was observed to decrease lesion size, as well as the number of parasites, and produced a higher IL-10 production and decrease IFN-γ secretion. Together, these results indicate that Hsp65-producing L. lactis can be considered as an alternative candidate for treatment in both autoimmune diseases, as well as in chronic infections that cause inflammatory disease.

Challenges in clinical-pathologic correlations: acute tubular necrosis in a patient with collapsing focal and segmental glomerulosclerosis mimicking rapidly progressive glomerulonephritis.

Herein, we report a case of acute kidney injury (AKI) due to diarrhea-induced acute tubular necrosis (ATN) in a patient with nephrotic syndrome secondary to biopsy-proven collapsing focal and segmental glomerulosclerosis (FSGS). The clinical picture mimicked rapidly progressive glomerulonephritis (RPGN) and motivated pulse therapy with methylprednisolone and cyclophosphamide. The case presentation is followed by a brief overview of the epidemiology of AKI in nephrotic syndrome as well as a discussion of its risk factors and potential mechanisms involved.

Upregulation of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in renal tissue in severe dengue in humans: Effects on endothelial activation/dysfunction.

INTRODUCTION: Dengue is an important mosquito-borne disease in tropical and subtropical regions. Adhesion molecules have not been systematically characterized in the renal tissue of patients with severe dengue (SD). The objective of this study was to detect viral antigens in samples from patients that evolved with SD, correlating with the expression of ICAM-1, VCAM-1, VE-cadherin, and E-selectin to contribute to a better understanding of the pathophysiology of SD. METHODS: Kidney specimens from patients with SD were selected according to clinical and laboratorial data and submitted to histological and immunohistochemistry analysis. A semiquantitative evaluation was performed considering positive immunostaining in 20 glomeruli. RESULTS: Viral antigens were mainly detected in distal tubules. The intense immunostaining of VCAM-1 and ICAM-1 was observed. The expression of E-selectin was discrete, and VE-cadherin expression varied from mild to moderate. VCAM-1 was slightly intense in the glomerular capsule; the expression of ICAM-1 was diffuse. E-selectin was diffuse, and VE-cadherin varied from mild to moderate. The most frequent histological findings were glomerular congestion, mild glomerulitis, acute renal injury, and glomerular atrophy. CONCLUSIONS: The results appear to demonstrate an imbalance between vascular endothelial permeability regulating events in renal lesions in SD. The increase in the expression of ICAM-1 and VCAM-1 is an in-situ indicator of higher permeability with a consequent influx of cells favoring the inflammation of the endothelium. These molecules are important in the pathophysiology of the disease and provide the possibility of developing new markers for the evaluation, clinical follow-up, and therapeutic response of patients with SD.

IGF-1 overexpression improves mesenchymal stem cell survival and promotes neurological recovery after spinal cord injury.

BACKGROUND: Survival and therapeutic actions of bone marrow-derived mesenchymal stem cells (BMMSCs) can be limited by the hostile microenvironment present during acute spinal cord injury (SCI). Here, we investigated whether BMMSCs overexpressing insulin-like growth factor 1 (IGF-1), a cytokine involved in neural development and injury repair, improved the therapeutic effects of BMMSCs in SCI. METHODS: Using a SCI contusion model in C57Bl/6 mice, we transplanted IGF-1 overexpressing or wild-type BMMSCs into the lesion site following SCI and evaluated cell survival, proliferation, immunomodulation, oxidative stress, myelination, and functional outcomes. RESULTS: BMMSC-IGF1 transplantation was associated with increased cell survival and recruitment of endogenous neural progenitor cells compared to BMMSC- or saline-treated controls. Modulation of gene expression of pro- and anti-inflammatory mediators was observed after BMMSC-IGF1 and compared to saline- and BMMSC-treated mice. Treatment with BMMSC-IGF1 restored spinal cord redox homeostasis by upregulating antioxidant defense genes. BMMSC-IGF1 protected against SCI-induced myelin loss, showing more compact myelin 28 days after SCI. Functional analyses demonstrated significant gains in BMS score and gait analysis in BMMSC-IGF1, compared to BMMSC or saline treatment. CONCLUSIONS: Overexpression of IGF-1 in BMMSC resulted in increased cell survival, immunomodulation, myelination, and functional improvements, suggesting that IGF-1 facilitates the regenerative actions of BMMSC in acute SCI.

IgA Nephropathy in Salvador, Brazil. Clinical and laboratory presentation at diagnosis.

INTRODUCTION: IgA nephropathy (IgAN) is the most prevalent primary glomerulopathy in the world, but great variation is reported in different countries. In Brazil, the reported prevalence is high in the Southeastern States and low in Salvador, Bahia State, Brazil. OBJECTIVES: This study investigated the clinical and histological patterns of patients with IgAN in Salvador, Brazil. METHODS: This is a descriptive study that included all patients with a diagnosis of IgAN performed in native kidney biopsies collected from referral nephrology services of public hospitals in Salvador between 2010 and 2015. Results: Thirty-two cases of IgAN were identified, corresponding to 6% of primary glomerulopathies. There was a slight male predominance (56%) and the median age was 30 [22-40] years. Hematuria was present in 79%, non-nephrotic proteinuria was present in 61%, and hypertension was present in 69% of patients. Segmental sclerosis (S1 lesions) was present in 81% of cases, and chronic tubulo-interstitial lesions (T1 and T2 lesions) were present in 44% of cases. Patients with M1 and T2 MEST-C scores exhibited higher serum urea and creatinine than other patients. CONCLUSION: The prevalence of IgAN was lower in Salvador than other regions of Brazil. Chronic histological lesions and laboratory markers of severe disease were frequent. M1 and T2 MEST-C scores were correlated with markers of renal dysfunction.

A monoclonal antibody against a canine CD45 homologue: analysis of tissue distribution, biochemical properties and in vitro immunological activity.

This report describes the characterisation of a monoclonal antibody (mAb), AB6, which recognises specifically a cluster of canine leukocyte surface molecules. The immunogen used for obtaining the AB6 mAb was a lysate of canine peripheral blood mononuclear cells (PBMC). This novel mAb belongs to the IgG2a isotype, and reacted in Western blot with four different canine leukocyte glycoproteins with apparent molecular weights of 180, 190, 205 and 220 kDa. The AB6 mAb recognised the majority of canine peripheral blood leukocytes as determined by flow cytometry (97%). It also exhibited a broad reactivity pattern against lymphoid and myeloid cells, inhibited the proliferation of mitogen-stimulated canine PBMC and did not recognise human PBMC and murine splenocytes. The biochemical properties, cell and tissue specificity, and in vitro biological activity of the AB6 mAb indicate that it recognises a canine CD45 homologue. The mAb could become a valuable diagnostic and research tool for the evaluation of immune functions in dogs.

Clinical and immunopathological findings during long term follow-up in Leishmania infantum experimentally infected dogs.

Canine Visceral Leishmaniasis (CVL) is caused by Leishmania infantum, which in the New World is transmitted by Lutzomyia longipalpis. While prospective clinical and immunological assessments of dogs experimentally challenged with L. infantum have been previously reported over a relatively short follow-up period, the long-term characterization of infected animals has not been performed to date. We evaluated dogs in a subclinical state for six years following experimental infection with L. infantum and Lu. longipalpis saliva, via an intradermal route, to characterize clinical, parasitological and immunological parameters arising from L. infantum experimental infection. We also assess these parameters in a group of naturally infected animals. The immune profiles of the experimentally and naturally infected animals exhibited increases of IFN-γ, IL-6 and IL-18, and decreases in TNF, IL-2, IL-8 and CXCL1, compared to controls. Our results indicate that over a six-year follow-up post-challenge, subclinically infected dogs presented low CVL clinical scores despite the persistence of Leishmania parasites in the lymph nodes, spleen and skin. Similarities observed among immune profiles in the context of experimental and natural infection seem to suggest that an enduring activation of the host immune response may lead to the control of parasite growth, thereby limiting disease severity.

Current distribution pattern of biopsy-proven glomerular disease in Salvador, Brazil, 40 years after an initial assessment.

INTRODUCTION: A report on the prevalence of glomerular disease diagnosed via renal biopsy in Salvador, BA, Brazil was published in 1973 and showed a predominance of membranoproliferative glomerulonephritis, which was frequently associated with hepatosplenic schistosomiasis. OBJECTIVE: In this study, we investigate the potential changes in the distribution of glomerular diseases after a period of important epidemiological transition in Brazil. METHODS: Pathology reports of all patients subjected to kidney biopsy from 2003 to 2015 in a referral nephrology service were reviewed. Clinical, laboratorial and pathological diagnoses were collected for analysis. Histological slides of the biopsies performed between 2003 and 2006 were reviewed to examine the accuracy of the estimates based on the pathology reports. RESULTS: Among the biopsies performed during the time period, 1,312 met the inclusion criteria for the study. Focal and segmental glomerulosclerosis was the most prevalent diagnosis, followed by lupus nephritis. However, a trend toward a decrease in the prevalence of focal and segmental glomerulosclerosis was detected (p < 0.05), and an increase in lupus (p < 0.0001) and membranous glomerulonephritis (p < 0.005) was observed. CONCLUSION: The data presented herein suggest the occurrence of changes in the distribution of nephrological diseases in Salvador, Brazil. The disease that was most prevalent shifted from membranoproliferative glomerulonephritis to focal and segmental glomerulosclerosis from 1975 to 2006 and from focal and segmental glomerulosclerosis to lupus nephritis from 2006 to 2015.

A standardized cytological and immunochemical method for the analysis of fine-needle spleen aspirates: assessment of leukocyte population changes in canine visceral leishmaniosis.

A method for the evaluation of splenic cellularity using samples collected by fine-needle aspirative biopsy was standardized in this work. The procedure includes erythrocyte lysing, preparation of cytospin films and staining by histochemical and immunocytochemical techniques. The cellular profiles of spleen preparations were compared with those observed in peripheral blood samples subjected to the same procedure. Two groups were compared, one consisting of 14 healthy uninfected and the other of 15 polysymptomatic Leishmania chagasi/infantum-infected dogs, from an endemic area for visceral leishmaniosis. Cell populations were identified by conventional hematoxilin-eosin and Wright' stainings, and by immunocytochemistry using monoclonal antibodies against canine CD45RA and CD45RB, phagocytes and a pan-leukocyte antigen. Larger neutrophil (P < 0.0001) and monocyte/macrophage (P = 0.0036) relative counts and lower lymphocyte relative counts (P < 0.0001) were found in the spleen, and not in the blood, of the animals with leishmaniosis than in those of the healthy animals. The proportions of CD45RB+ cells were higher, and of CD45RA+ cells were lower, both in the spleen and in the blood of animals with leishmaniosis than in those of healthy dogs (P < 0.05). Additionally, hematoxilin-eosin-stained cytospins of spleen aspirates from Leishmania-infected animals permitted the easy visualization of amastigote forms inside phagocytes, under light microscopy.

Is there a relationship between the detection of human herpesvirus 8 and Epstein-Barr virus in Waldeyer's ring tissues?

OBJECTIVE: Human herpesvirus 8 (HHV-8) and Epstein-Barr virus (EBV) are human pathogens associated to a number of neoplasms, including tumors of the Waldeyer's ring. Both viruses have been previously detected by in situ methods in tonsils and adenoids from children. HHV-8 was found in 6.8% of the cases and EBV in about one third of the cases. As they belong to the same gamma-herpesvirus subfamily and share some biological characteristics, it is of medical interest to further explore their possible relationship in the Waldeyer's ring, an issue not yet addressed in the specialized literature. The purpose of the present study is to compare the presence of EBV by in situ hybridization (ISH) in tonsils and adenoids from children up to 14 years of age in cases previously shown to be positive and negative for HHV-8. METHODS: Paraffin wax-embedded sections consisting of 38 tonsils and two adenoids from 40 patients were analyzed. HHV-8 was detected by ISH, using the T1-1 probe for the viral mRNA. EBV was also detected by ISH, using the EBER probe. Both probes and the detection systems were provided by Novocastra. RESULTS: HHV-8 was detected in 19 tonsils and one adenoid. The other 19 tonsils and one adenoid taken from the HHV-8-negative group were selected by pairing age and gender of patients with the HHV-8-positive group. In both groups EBV was detected in 13 cases and was negative in other 7. CONCLUSION: Although both viruses are related in many aspects, some biological and epidemiological features differ. This is reflected in the present results, as EBV is similarly detected in the groups negative and positive for HHV-8, favoring different mechanisms of spread.

Recombinant single-chain canine interleukin 12 induces interferon gamma mRNA expression in peripheral blood mononuclear cells of dogs with visceral leishmaniasis.

Canine visceral leishmaniasis poses important concerns for public health and veterinary medicine in many areas of the world. Resistance to it seems to be associated with cellular specific immune responses of the so-called Th1 type. Interleukin-12 (IL-12) is one of the most potent inducers of Th1 type of immune responses to co-administered antigens. Herein, the cloning of canine IL-12, as a single-chain fusion protein (sccaIL-12), and its expression in biologically active form in COS-7 cells is reported. Supernatants from these cells stimulated the expression of comparable amounts of interferon gamma mRNA in peripheral blood mononuclear cells from dogs with natural visceral leishmaniasis. In addition, after stimulation with sccaIL-12, there was no difference between interferon gamma mRNA expressions in peripheral blood mononuclear cells of dogs with visceral leishmaniasis and from normal healthy control animals.