Unraveling the mystery of Gaucher bone density pathophysiology.

Gaucher disease (GD) is caused by pathogenic mutations in GBA1, the gene that encodes the lysosomal enzyme ß-glucocerebrosidase. Despite the existence of a variety of specific treatments for GD, they cannot completely reverse bone complications. Many studies have evidenced the impairment in bone tissue of GD, and molecular mechanisms of bone density alterations in GD are being studied during the last years and different reports emphasized its efforts trying to unravel why and how bone tissue is affected. The cause of skeletal density affection in GD is a matter of debates between research groups. and there are two opposing hypotheses trying to explain reduced bone mineral density in GD: increased bone resorption versus impaired bone formation. In this review, we discuss the diverse mechanisms of bone alterations implicated in GD revealed until the present, along with a presentation of normal bone physiology and its regulation. With this information in mind, we discuss effectiveness of specific therapies, introduce possible adjunctive therapies and present a novel model for GD-associated bone density pathogenesis. Under the exposed evidence, we may conclude that both sides of the balance of remodeling process are altered. In GD the observed osteopenia/osteoporosis may be the result of contribution of both reduced bone formation and increased bone resorption.

Gaucher disease-associated alterations in mesenchymal stem cells reduce osteogenesis and favour adipogenesis processes with concomitant increased osteoclastogenesis.

Gaucher disease (GD) is caused by pathogenic mutations in GBA1, the gene that encodes the lysosomal enzyme ß-glucocerebrosidase. Until now, treatments for GD cannot completely reverse bone problems. The aim of this work was to evaluate the potential of MSCs from GD patients (GD MSCs) to differentiate towards the osteoblast (GD Ob) and adipocyte (GD Ad) lineages, and their role in osteoclastogenesis. We observed that GD Ob exhibited reduced mineralization, collagen deposition and alkaline phosphatase activity (ALP), as well as decreased gene expression of RUNX2, COLA1 and ALP. We also evaluated the process of osteoclastogenesis and observed that conditioned media from GD MSCs supernatants induced an increase in the number of osteoclasts. In this model, osteoclastogenesis was induced by RANKL and IL-1ß. Furthermore, results showed that in GD MSCs there was a promotion in NLRP3 and PPAR-γ gene expression. Adipogenic differentiation revealed that GD Ad had an increase in PPAR-γ and a reduced RUNX2 gene expression, promoting adipocyte differentiation. In conclusion, our results show that GD MSCs exhibited deficient GD Ob differentiation and increased adipogenesis. In addition, we show that GD MSCs promoted increased osteoclastogenesis through RANKL and IL-1ß. These changes in GD MSCs are likely to contribute to skeletal imbalance observed in GD patients.

Proinflammatory and proosteoclastogenic potential of peripheral blood mononuclear cells from Gaucher patients: Implication for bone pathology.

Gaucher disease (GD) is caused by mutations in the GBA gene that confer a deficient level of activity of glucocerebrosidase (GCase). This deficiency leads to the accumulation of the glycolipid glucocerebroside in the lysosomes of cells of monocyte/macrophage system. Bone compromise in Gaucher disease patients is the most disabling aspect of the disease. However, pathophysiological aspects of skeletal alterations are still poorly understood. On the other hand it is well known that inflammation is a key player in GD pathology. In this work, we revealed increased levels of the proinflammatory CD14(+)CD16(+) monocyte subset and increased inflammatory cytokine production by monocytes and T cells in the circulation of GD patients. We showed increased levels of osteoclast precursors in PBMC from patients and a higher expression of RANKL in the surface of T cells. PBMC from patients presented higher osteoclast differentiation compared to healthy controls when cultured in the presence of M-CSF alone or in combination with RANKL. In vitro treatment with Velaglucerase reduced osteoclast levels to control levels. On the other hand THP-1 derived osteoclast precursors cultured in the presence of conditioned media from PBMC of GD patients presented higher differentiation to active osteoclasts. This induction involved TNF-α and RANKL.

Bone marrow adipocytes alteration in an in vitro model of Gaucher Disease.

Gaucher disease (GD) is caused by biallelic pathogenic variants in GBA1 gene that encodes the lysosomal enzyme glucocerebrosidase. Up to now, specific treatment for GD cannot completely reverse bone complications. Bone is composed of different cell types; including osteoblasts, osteocytes and osteoclasts. Osteoblasts are present on bone surfaces and are derived from local mesenchymal stem cells (MSCs). Depending on environment conditions, MSCs could differentiate into osteoblasts and adipocytes. Mature adipocytes-secreted adipokines and free fatty acids affect both osteoblasts and osteoclasts formation/activity and therefore mediate skeletal homeostasis. The aim of this study was to evaluate possible alterations in GD adipocyte (GD Ad) that could contribute to bone complications. MSCs were grown in adipogenic media in order to evaluate expression of differentiation markers as PPAR-γ. PPAR-γ was observed into the nucleus of GD Ad, indicating that these cells are properly stimulated. However, these cells accumulate lesser lipid droplets (LDs) than Control Ad. In order to study lipid droplet metabolism, we evaluated the lipolysis of these structures by the measurement of free glycerol in culture supernatant. Our results indicated that GD Ad had an alteration in this process, evidenced by an increase in glycerol release. We have also evaluated two enzymes involved in LDs synthesis: fatty acid synthase (FASN) and stearoyl-coenzyme A desaturase 1 (SCD1). The transcription of these genes was decreased in GD Ad, suggesting a dysfunction in the synthesis of LDs. In conclusion, our results show an alteration in LDs metabolism of GD Ad, independent of adipocyte differentiation process. This alteration would be caused by an increase in lipolysis in early stages of differentiation and also by a reduction of lipid synthesis, which could contribute with the skeletal imbalance in GD.

In vitro osteoclastogenesis from Gaucher patients' cells correlates with bone mineral density but not with Chitotriosidase.

Gaucher disease (GD) is caused by mutations on the gene encoding for the lysosomal enzyme glucocerebrosidase. Type I GD (GD1) patients present anemia, hepatosplenomegaly and bone alterations. In spite of treatment, bone alterations in GD patients persist, including poor bone mineral density (BMD). Mechanisms leading to bone damage are not completely understood, but previous reports suggest that osteoclasts are involved. Chitotriosidase (CHIT) is the most reliable biomarker used in the follow up of patients, although its correlation with bone status is unknown. The aim of this work was to study the pro-osteoclastogenic potential in patients and to evaluate its correlation with CHIT activity levels and clinical parameters. PBMCs from treated patients and healthy controls were cultured in the presence of M-CSF, and mature osteoclasts were counted. BMD, blood CHIT activity and serum levels of CTX, BAP, and cytokines were evaluated in patients. We found that blood CHIT activity and osteoclast differentiation were significantly increased in patients, but no correlation between them was observed. Interestingly, osteoclast numbers but not CHIT, presented a negative correlation with BMD expressed as Z-score. CTX, BAP and serum cytokines involved in bone remodeling were found altered in GD1 patients. These results show for the first time a correlation between osteoclast differentiation and BMD in GD1 patients, supporting the involvement of osteoclasts in the bone pathology of GD1. Our results also suggest that an altered immune response may play an important role in bone damage.

Giardia and Cryptosporidium in source waters of São Paulo State, Brazil.

Giardia lamblia and Cryptosporidium parvum are two protozoan intestinal parasites responsible for many drinking-water-related disease outbreaks in recent years. They are very resistant to conventional water treatment processes, can persist for long times in the environment and are, therefore, of great concern for public health. This work aimed to evaluate the presence of Giardia and Cryptosporidium in water sources from São Paulo State, Brazil, as part of the "Evaluation of Inland Waters from São Paulo State" project from CETESB. Over a period of 19 months, 278 water samples from 28 sites located in 10 watersheds were analysed. The immunofluorescence assay was used after concentration of the samples by the calcium carbonate flocculation technique. Thermotolerant (faecal) coliforms, faecal streptococci and Clostridium perfringens were also determined in order to verify the existence of correlation between these bacterial indicators and the protozoa. Giardia and Cryptosporidium were detected in 27% and 2.5% of the samples, respectively, a lower figure compared with the results reported by other authors, especially for Cryptosporidium. A Spearman rank correlation test demonstrated a significant correlation between Giardia and faecal indicator concentrations. According to the American Regulation of Monitoring (ICR), treated water from 16 of these 28 collection sites should also be analysed to evaluate whether the treatment process could remove the parasites. Some technical deficiencies of these methods still limit the utilisation of the monitoring results for public health decisions, but the data here reported will help to improve the quality of drinking water in São Paulo State.

Population dynamics: seasonal variation of phytoplankton functional groups in brazilian reservoirs (Billings and Guarapiranga, São Paulo).

Phytoplankton may function as a 'sensor' of changes in aquatic environment and responds rapidly to such changes. In freshwaters, coexistence of species that have similar ecological requirements and show the same environmental requirements frequently occurs; such species groups are named functional groups. The use of phytoplankton functional groups to evaluate these changes has proven to be very useful and effective. Thus, the aim of this study was to evaluate the occurrence of functional groups of phytoplankton in two reservoirs (Billings and Guarapiranga) that supply water to millions of people in São Paulo city Metropolitan Area, southeastern Brazil. Surface water samples were collected monthly and physical, chemical and biological (quantitative and qualitative analyses of the phytoplankton) were performed. The highest biovolume (mm(3).L-1) of the descriptor species and functional groups were represented respectively by Anabaena circinalis Rabenh. (H1), Microcystis aeruginosa (Kützing) Kützing (L M/M) and Mougeotia sp. (T) in the Guarapiranga reservoir and Cylindrospermopsis raciborskii (Wolosz.) Seen. and Subba Raju (S N), Microcystis aeruginosa and M. panniformis Komárek et al. (L M/M), Planktothrix agardhii (Gom.) Anagn. and Komárek and P. cf. clathrata (Skuja) Anagn. and Komárek (S1) in the Billings reservoir. The environmental factors that most influenced the phytoplankton dynamics were water temperature, euphotic zone, turbidity, conductivity, pH, dissolved oxygen, nitrate and total phosphorous.

Estudo do inseticida carbofurano em solo e sedimento de área de produção de arroz irrigado e controle do gorgulho aquático Oryzophagus oryzae, Taubaté, São Paulo, Brasil / Study of the insecticide carbofuran in the soil and sediment of paddy rice fields with control of the weevil Oryzophagus oryzae, in Taubaté, state of São Paulo, Brazil

Resíduos do inseticida e nematicida carbofurano e de seu principal metabólito 3-hidroxicarbofurano foram averiguados em tabuleiros de arroz irrigado e em áreas adjacentes para controle do gorgulho aquático da espécie Oryzophagus oryzae Lima, 1936. As amostras foram coletadas no período de 6/9/1999 a 4/5/2000 e analisadas por cromatografia a líquido de alta eficiência (HPLC) utilizando-se detector de fluorescência. Não foram detectados resíduos de carbofurano e de seu metabólito 3-hidroxicarbofurano dentro dos limites de determinação do método.

The presence of residues of the insecticide and nematicide carbofuran, used for weevil (Oryzophagus oryzae Lima 1936) control, and its principal metabolite 3-hydroxycarbofuran were evaluated in soil and sediment plots from paddy rice fields and adjacent areas. The samples were analyzed using a high-performance liquid chromatography and fluorescence detector. Carbofuran and 3-hydroxycarbofuran residues were not detected in any sample in amounts above the method’s detection threshold.

The kinetic energy change on covalent bond formation.

Stimulated by an analysis of the classical molecular orbital and valence bond descriptions of the two-electron normal covalent bond (both faulty), the argument is made that there exist good representations of the kinetic energy change DeltaT, on nonpolar covalent bond formation in a diatomic molecule, of the form DeltaT(R) = integralF(R - r')S(r')dr'. Here F is a nonlinear response function which itself involves the overlap S. The kinetic change is known to satisfy the sum rule integral(0) (infinity)DeltaT(R)dR = Z(alpha)Z(beta) exactly; it is shown how this can be built into the treatment by the use of Fourier transform methods. Also considered is integral(0) (infinity)DeltaT(R)R(2)dR, which is an important additional property of the kinetic energy change. Representation of DeltaT(R) as a Morse function, already known to be highly accurate, is shown to exactly conform to the proposed form.

Epitope mapping of trans-sialidase from Trypanosoma cruzi reveals the presence of several cross-reactive determinants.

Trypanosoma cruzi, the agent of Chagas' disease, expresses trans-sialidase, a unique enzyme activity that enables the parasite to invade host cells by transferring sialyl residues from host glyconjugates to the parasite's surface acceptor molecules. The enzyme is also shed into the surrounding environment, causing apoptosis in cells from the immune system. During infections, an antibody response against the catalytic region of the trans-sialidase that is coincident with the control of the parasitemia and survival of the host is observed. This low-titer humoral response is characterized by its persistence for many years in benznidazole-treated patients. Here we analyzed the antigenic structure of the molecule by phage-displayed peptide combinatorial libraries and SPOT synthesis. Several epitopes were defined and located on the three-dimensional model of the enzyme. Unexpectedly, cross-reaction was found among several epitopes distributed in different locations displaying nonconsensus sequences. This finding was confirmed by the reactivity of three monoclonal antibodies able to recognize non-sequence-related peptides that together constitute the surface surrounding the catalytic site of the enzyme. The presence of cross-reacting epitopes within a single molecule suggests a mechanism developed to avoid a strong humoral response by displaying an undefined target to the immune system.

Alpha-difluoromethylornithine-resistant cell lines obtained after one-step selection of Leishmania mexicana promastigote cultures.

Proliferation of Leishmania mexicana promastigotes in synthetic medium can be blocked by the depletion of intracellular polyamine pools induced by the presence of D,L-alpha-difluoromethylornithine (DFMO), a specific and irreversible inhibitor of ornithine decarboxylase (ODC). Here we report that DFMO-resistant cell lines growing normally at DFMO levels of 10 mM have been obtained from non-proliferating cultures after a single-step selection in the presence of high concentrations of the drug. The DFMO-resistant promastigotes underwent a morphological transformation into an 'amastigote-like' form after incubation for several hours at gradually increasing temperatures up to 35 degrees C. The uptake of DFMO was not significantly altered in the drug-resistant cell lines but in both cases (promastigote and 'amastigote-like' forms) the ODC specific activity was increased approx. 15-fold over the normal enzymic levels found in the wild-type Leishmania. The enzyme affinities for its substrate and for DFMO gave very similar values in the drug-resistant promastigotes and the wild-type parasites. In contrast, ODC from the 'amastigote-like' Leishmania showed a higher affinity for ornithine and a decreased capacity for the binding of DFMO. An 80-fold amplification of the ODC gene and a corresponding increase in its transcripts have been detected in both DFMO-resistant Leishmania cell lines. The drug-resistant phenotypes with their characteristic morphologies, the increased levels of ODC activity and the amplification of the ODC gene have been stable for at least 6 months in the absence of selective pressure.