Bronchoalveolar lavage as a tool for evaluation of cellular alteration during Aelurostrongylus abstrusus infection in cats

Bronchoalveolar lavage (BAL) is a procedure that retrieves cells and other elements from the lungs for evaluation, which helps in the diagnosis of pulmonary diseases. The aim of this study was to perform this procedure for cellular analysis of BAL fluid alterations during experimental infection with Aelurostrongylus abstrusus in cats. Fourteen cats were individually inoculated with 800 third stage larvae of A. abstrusus and five non-infected cats lined as a control group. The BAL procedure was performed through the use of an endotracheal tube on the nineteen cats with a mean age of 18 months, on 0, 30, 60, 90, 120, 180 and 270 days after infection. Absolute cell counts in the infected cats revealed that alveolar macrophages and eosinophils were the predominant cells following infection. This study shows that the technique allows us to retrieve cells and first stage larvae what provides information about the inflammatory process caused by aelurostrongylosis.

Systemic acanthamoebiasis associated with canine distemper in dogs in the semiarid region of Paraíba, Brazil

Infections by free-living amoebae can cause systemic disease in animals and humans. We describe the epidemiological, clinical and pathological aspects of disseminated acanthamoebiasis associated with canine distemper in three dogs of the semiarid region of Paraíba, Northeastern Brazil. Affected dogs developed progressive neurological and respiratory signs that progressed to death within in two to 20 days. Gross lesions were irregular and with yellow-reddish nodules randomly distributed in the lungs, heart, kidneys, spleen, lymph nodes, adrenals, and intestine. One dog had foci of malacia in the parietal cortex and another one in nucleus of brain basis. Histologically, pyogranulomas with areas of necrosis and hemorrhage in all organs affected were observed, associated with myriads of intralesional amoebic trophozoites. All three cases were concomitant canine distemper, that possibly triggered immunosuppression in the dogs. The diagnosis was performed through microscopic findings of infection by free-living amoebae and confirmed Acanthamoeba sp. by immunohistochemistry

Markers of Bone Turnover In Preclinical Development of Drugs for Skeletal Diseases

Skeletal tissue is constantly remodeled in a process where osteoclasts resorb old bone and osteoblasts form new bone. Balance in bone remodeling is related to age, gender and genetic factors, but also many skeletal diseases, such as osteoporosis and cancer-induced bone metastasis, cause imbalance in bone turnover and lead to decreased bone mass and increased fracture risk. Biochemical markers of bone turnover are surrogates for bone metabolism and may be used as indicators of the balance between bone resorption and formation. They are released during the remodeling process and can be conveniently and reliably measured from blood or urine by immunoassays. Most commonly used bone formation markers include N-terminal propeptides of type I collagen (PINP) and osteocalcin, whereas tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) and C-terminal cross-linked telopeptide of type I collagen (CTX) are common resorption markers. Of these, PINP has been, until recently, the only marker not commercially available for preclinical use. To date, widespread use of bone markers is still limited due to their unclear biological significance, variability, and insufficient evidence of their prognostic value to reflect long term changes. In this study, the feasibility of bone markers as predictors of drug efficacy in preclinical osteoporosis models was elucidated. A non-radioactive PINP immunoassay for preclinical use was characterized and validated. The levels of PINP, N-terminal mid-fragment of osteocalcin, TRACP 5b and CTX were studied in preclinical osteoporosis models and the results were compared with the results obtained by traditional analysis methods such as histology, densitometry and microscopy. Changes in all bone markers at early timepoints correlated strongly with the changes observed in bone mass and bone quality parameters at the end of the study. TRACP 5b correlated strongly with the osteoclast number and CTX correlated with the osteoclast activity in both in vitro and in vivo studies. The concept “resorption index” was applied to the relation of CTX/TRACP 5b to describe the mean osteoclast activity. The index showed more substantial changes than either of the markers alone in the preclinical osteoporosis models used in this study. PINP was strongly associated with bone formation whereas osteocalcin was associated with both bone formation and resorption. These results provide novel insight into the feasibility of PINP, osteocalcin, TRACP 5b and CTX as predictors of drug efficacy in preclinical osteoporosis models. The results support clinical findings which indicate that short-term changes of these markers reflect long-term responses in bone mass and quality. Furthermore, this information may be useful when considering cost-efficient and clinically predictive drug screening and development assays for mining new drug candidates for skeletal diseases.

The role of cathepsin K in lung development and newborn chronic lung injury.

Chronic lung diseases, specifically bronchopulmonary dysplasia (BPD), are still causing mortality and morbidity amongst newborn infants. High protease activity has been suggested to have a deleterious role in oxygen-induced lung injuries. Cathepsin K (CatK) is a potent protease found in fetal lungs, degrading collagen and elastin. We hypothesized that CatK may be an important modulator of chronic lung injury in newborn infants and neonatal mice. First we measured CatK protein levels in repeated tracheal aspirate fluid samples from 13 intubated preterm infants during the first two weeks of life. The amount of CatK at 9-13 days was low in infants developing chronic lung disease. Consequently, we studied CatK mRNA expression in oxygen-exposed wild-type (WT) rats at postnatal day (PN) 14 and found decreased pulmonary mRNA expression of CatK in whole lung samples. Thereafter we demonstrated that CatK deficiency modifies lung development by accelerating the thinning of alveolar walls in newborn mice. In hyperoxia-exposed newborn mice CatK deficiency resulted in increased number of pulmonary foam cells, macrophages and amount of reduced glutathione in lung homogenates indicating intensified pulmonary oxidative stress and worse pulmonary outcome due to CatK deficiency. Conversely, transgenic overexpression of CatK caused slight enlargement of distal airspaces with increased alveolar chord length in room air in neonatal mice. While hyperoxic exposure inhibited alveolarization and resulted in enlarged airspaces in wild-type mice, these changes were significantly milder in CatK overexpressing mice at PN7. Finally, we showed that the expression of macrophage scavenger receptor 2 (MSR2) mRNA was down-regulated in oxygen-exposed CatK-deficient mice analyzed by microarray analysis. Our results demonstrate that CatK seems to participate in normal lung development and its expression is altered during pulmonary injury. In the presence of pulmonary risk factors, like high oxygen exposure, low amount of CatK may contribute to aggravated lung injury while sustained or slightly elevated amount of CatK may even protect the newborn lungs from excessive injury. Besides collagen degrading and antifibrotic function of CatK in the lungs, it is obvious that CatK may affect macrophage activity and modify oxidative stress response. In conclusion, pulmonary proteases, specifically CatK, have distinct roles in lung homeostasis and injury development, and although suggested, broad range inhibition of proteases may not be beneficial in newborn lung injury.

Human DNA repair diseases: From genome instability to cancer

Several human genetic syndromes have long been recognized to be defective in DNA repair mechanisms. This was first discovered by Cleaver (1968), who showed that cells from patients with xeroderma pigmentosum (XP) were defective for the ability to remove ultraviolet (UV)-induced lesions from their genome. Since then, new discoveries have promoted DNA repair studies to one of the most exciting areas of molecular biology. The present work intends to give a brief summary of the main known human genetic diseases related to DNA repair and how they may be linked to acquired diseases such as cancer

Assessment of ventilatory neuromuscular drive in patients with obstructive sleep apnea

The presence of abnormalities of the respiratory center in obstructive sleep apnea (OSA) patients and their correlation with polysomnographic data are still a matter of controversy. Moderately obese, sleep-deprived OSA patients presenting daytime hypersomnolence, with normocapnia and no clinical or spirometric evidence of pulmonary disease, were selected. We assessed the ventilatory control and correlated it with polysomnographic data. Ventilatory neuromuscular drive was evaluated in these patients by measuring the ventilatory response (VE), the inspiratory occlusion pressure (P.1) and the ventilatory pattern (VT/TI, TI/TTOT) at rest and during submaximal exercise, breathing room air. These analyses were also performed after inhalation of a hypercapnic mixture of CO2 (DP.1/DPETCO2, DVE/DPETCO2). Average rest and exercise ventilatory response (VE: 12.2 and 32.6 l/min, respectively), inspiratory occlusion pressure (P.1: 1.5 and 4.7 cmH2O, respectively), and ventilatory pattern (VT/TI: 0.42 and 1.09 l/s; TI/TTOT: 0.47 and 0.46 l/s, respectively) were within the normal range. In response to hypercapnia, the values of ventilatory response (DVE/DPETCO2: 1.51 l min-1 mmHg-1) and inspiratory occlusion pressure (DP.1/DPETCO2: 0.22 cmH2O) were normal or slightly reduced in the normocapnic OSA patients. No association or correlation between ventilatory neuromuscular drive and ventilatory pattern, hypersomnolence score and polysomnographic data was found; however a significant positive correlation was observed between P.1 and weight. Our results indicate the existence of a group of normocapnic OSA patients who have a normal awake neuromuscular ventilatory drive at rest or during exercise that is partially influenced by obesity

Glycosuria in glomerular diseases: histopathology and clinical correlations

There are doubts about the presence of glycosuria and the progress of glomerular disease. Some reports suggest that glycosuria could be an index of a more severe tubulointerstitial lesion. We investigated the presence of glycosuria in 60 patients with primary glomerular diseases: 17 patients (28%) had glycosuria and 43 patients (72%) were glycosuria free. The two groups were similar in age, arterial pressure and sex. Serum creatinine was higher in patients with glycosuria (2.0 ± 1.7 vs 1.3 ± 0.9 mg/dl, P<0.05). The protein excretion rate was 7.5 ± 3.7 vs 5.3 ± 4.2 g/day (P>0.05) in patients with and without glycosuria, respectively, while serum albumin was lower in patients with glycosuria (1.7 ± 0.6 vs 2.7 ± 1.0 g/dl, P<0.05). Several histological forms were present in the group with glycosuria, with membranous glomerulonephritis being the most frequent. Histological evidence of tubular atrophy and interstitial fibrosis prevailed in patients with glycosuria, suggesting a poor prognosis for these patients. We may conclude that the presence of glycosuria in patients with glomerular disease is associated with more pronounced tubular atrophy and interstitial fibrosis and therefore imply a poorer prognosis.

DNA vaccines for viral diseases

DNA plasmids encoding foreign proteins may be used as immunogens by direct intramuscular injection alone, or with various adjuvants and excipients, or by delivery of DNA-coated gold particles to the epidermis through biolistic immunization. Antibody, helper T lymphocyte, and cytotoxic T lymphocyte (CTL) responses have been induced in laboratory and domesticated animals by these methods. In a number of animal models, immune responses induced by DNA vaccination have been shown to be protective against challenge with various infectious agents. Immunization by injection of plasmids encoding foreign proteins has been used successfully as a research tool. This review summarizes the types of DNA vaccine vectors in common use, the immune responses and protective responses that have been obtained in animal models, the safety considerations pertinent to the evaluation of DNA vaccines in humans and the very limited information that is available from early clinical studies.

Apoptosis in parasites and parasite-induced apoptosis in the host immune system: a new approach to parasitic diseases

Apoptosis, a form of programmed cell death (PCD), has been described as essential for normal organogenesis and tissue development, as well as for the proper function of cell-renewal systems in adult organisms. Apoptosis is also pivotal in the pathogenesis of several different diseases. In this paper we discuss, from two different points of view, the role of apoptosis in parasitic diseases. The description of apoptotic death in three different species of heteroxenic trypanosomatids is reviewed, and considerations on the phylogenesis of apoptosis and on the eventual role of PCD on their mechanism of pathogenesis are made. From a different perspective, an increasing body of evidence is making clear that regulation of host cell apoptosis is an important factor on the definition of a host-pathogen interaction. As an example, the molecular mechanisms by which Trypanosoma cruzi is able to induce apoptosis in immunocompetent cells, in a murine model of Chagas' disease, and the consequences of this phenomenon on the outcome of the experimental disease are discussed.

Reactivity of anti-thyroid antibodies to thyroglobulin tryptic fragments: comparison of autoimmune and non-autoimmune thyroid diseases

Studies concerning the antigenicity of thyroglobulin fragments allow the characterization of the epitopes but do not consider the role of heavier antigenic fragments that could result in vivo from the action of endoproteases. Here we assess the relative importance of the fragments obtained from thyroglobulin by limited proteolysis with trypsin and compare by immunoblotting their reactivity to serum from patients with autoimmune (Graves' disease and Hashimoto's thyroiditis) and non-autoimmune (subacute thyroiditis) disease. The results showed no difference in frequency of recognition of any peptide by sera from patients with autoimmune thyroiditis. In contrast, sera from patients with subacute thyroiditis reacted more frequently with a peptide of 80 kDa. These results suggest the presence of antibody subpopulations directed at fragments produced in vivo by enzymatic cleavage of thyroglobulin. This fragment and antibodies to it may represent markers for subacute thyroiditis.

Detection of specific IgE antibodies in parasite diseases

Activation of Th1 or Th2 cells is associated with production of specific immunoglobulin isotypes, offering the opportunity to use antibody measurement for evaluation of T cell function. Schistosomiasis and visceral leishmaniasis are diseases associated with Th2 activation. However, an IgE response is not always detected in these patients. In the present study we evaluated specific IgE antibodies to S. mansoni and L. chagasi antigens by ELISA after depletion of serum IgG with protein G immobilized on Sepharose beads or RF-absorbent (purified sheep IgG antibodies anti-human IgG). In schistosomiasis patients, specific IgE to SWAP antigen was demonstrable in only 10 of 21 patients (48%) (mean absorbance ± SD = 0.102 ± 0.195) when unabsorbed serum was used. Depletion of IgG with protein G increased the number of specific IgE-positive tests to 13 (62%) and the use of RF-absorbent increased the number of positive results to 20 (95%) (mean absorbances ± SD = 0.303 ± 0.455 and 0.374 ± 0.477, respectively). Specific IgE anti-L. chagasi antibodies were not detected in unabsorbed serum from visceral leishmaniasis patients. When IgG was depleted with protein G, IgE antibodies were detected in only 3 (11%) of 27 patients, and the use of RF-absorbent permitted the detection of this isotype in all 27 visceral leishmaniasis sera tested (mean absorbance ± SD = 0.104 ± 0.03). These data show that the presence of IgG antibodies may prevent the detection of a specific IgE response in these parasite diseases. RF-absorbent, a reagent that blocks IgG-binding sites and also removes rheumatoid factor, was more efficient than protein G for the demonstration of specific IgE antibodies.

Measurement of intestinal permeability using mannitol and lactulose in children with diarrheal diseases

The excretion ratio of lactulose/mannitol in urine has been used to assess the extension of malabsorption and impairment of intestinal permeability. The recovery of lactulose and mannitol in urine was employed to evaluate intestinal permeability in children with and without diarrhea. Lactulose and mannitol probes were measured using high-performance liquid chromatography with pulsed amperometric detection (HPLC-PAD). Two groups of solutions containing 60 µM sugars were prepared. Group I consisted of glucosamine, mannitol, melibiose and lactulose, and group II of inositol, sorbitol, glucose and lactose. In the study of intra-experiment variation, a sample of 50 µl from each group was submitted to 4 successive determinations. The recovered amounts and retention times of each sugar showed a variation <2 and 1%, respectively. The estimated recovery was >97%. In the study of inter-experiment variation, we prepared 4 independent samples from groups I and II at the following concentrations: 1.0, 0.3, 0.1, 0.03 and 0.01 mM. The amounts of the sugars recovered varied by <10%, whereas the retention times showed an average variation <1%. The linear correlation coefficients were >99%. Retention (k'), selectivity (a) and efficiency (N) were used to assess the chromatographic conditions. All three parameters were in the normal range. Children with diarrhea presented a greater lactulose/mannitol ratio compared to children without diarrhea.

Extracellular calcium-sensing receptor: structural and functional features and association with diseases

The recently cloned extracellular calcium-sensing receptor (CaR) is a G protein-coupled receptor that plays an essential role in the regulation of extracellular calcium homeostasis. This receptor is expressed in all tissues related to this control (parathyroid glands, thyroid C-cells, kidneys, intestine and bones) and also in tissues with apparently no role in the maintenance of extracellular calcium levels, such as brain, skin and pancreas. The CaR amino acid sequence is compatible with three major domains: a long and hydrophilic aminoterminal extracellular domain, where most of the activating and inactivating mutations described to date are located and where the dimerization process occurs, and the agonist-binding site is located, a hydrophobic transmembrane domain involved in the signal transduction mechanism from the extracellular domain to its respective G protein, and a carboxyterminal intracellular tail, with a well-established role for cell surface CaR expression and for signal transduction. CaR cloning was immediately followed by the association of genetic human diseases with inactivating and activating CaR mutations: familial hypocalciuric hypercalcemia and neonatal severe hyperparathyroidism are caused by CaR-inactivating mutations, whereas autosomal dominant hypoparathyroidism is secondary to CaR-activating mutations. Finally, we will comment on the development of drugs that modulate CaR function by either activating (calcimimetic drugs) or antagonizing it (calcilytic drugs), and on their potential therapeutic implications, such as medical control of specific cases of primary and uremic hyperparathyroidism with calcimimetic drugs and a potential treatment for osteoporosis with a calcilytic drug.

Scintigraphy and Doppler ultrasonography for the evaluation of obstructive urinary calculi

Forty-seven patients with unilateral obstructive calculi (12 males and 35 females) were submitted to 99mTc-diethylene triamine pentaacetic acid (DTPA) or 99mTc-dimercaptosuccinic acid (DMSA) scans for assessment of renal function. The scans revealed unilateral functional deficit in 68 and 66% of the patients, respectively. A calculus size of 1.1 to 2.0 cm was significantly associated with deficit detected by DTPA, but duration of obstruction and calculus localization were not. After relief of the obstruction, the mean percent renal function of the affected kidney was found to be significantly increased from 25 ± 12% to 29 ± 12% in DTPA and from 21 ± 15% to 24 ± 12% in DMSA. Initial Doppler ultrasonography performed in 35 patients detected an increased resistive index in 10 (29%). In the remaining patients with a normal resistive index, ureteral urinary jet was observed, indicating partial obstruction. The high frequency of renal function impairment detected by DTPA and of tubulointerstitial damage detected by DMSA as well as the slight amelioration of unilateral renal function after relief of obstruction suggest that scintigraphy assessment may help evaluate the unilateral percentage of renal function and monitor renal function recovery when it occurs. The presence of a urinary jet detected by Doppler ultrasonography further indicates the severity of obstruction and the recovery prognosis.

Establishment of new murine embryonic stem cell lines for the generation of mouse models of human genetic diseases

Embryonic stem cells are totipotent cells derived from the inner cell mass of blastocysts. Recently, the development of appropriate culture conditions for the differentiation of these cells into specific cell types has permitted their use as potential therapeutic agents for several diseases. In addition, manipulation of their genome in vitro allows the creation of animal models of human genetic diseases and for the study of gene function in vivo. We report the establishment of new lines of murine embryonic stem cells from preimplantation stage embryos of 129/Sv mice. Most of these cells had a normal karyotype and an XY sex chromosome composition. The pluripotent properties of the cell lines obtained were analyzed on the basis of their alkaline phosphatase activity and their capacity to form complex embryoid bodies with rhythmically contracting cardiomyocytes. Two lines, USP-1 and USP-3, with the best in vitro characteristics of pluripotency were used in chimera-generating experiments. The capacity to contribute to the germ line was demonstrated by the USP-1 cell line. This cell line is currently being used to generate mouse models of human diseases.