Efficacy of diadynamic currents as an adjunct to exercise to manage symptoms of knee osteoarthritis in adults: A randomized controlled clinical trial.

OBJECTIVE: To investigate the effect of diadynamic currents administered prior to exercises on pain and disability in patients with osteoarthritis of the knee. DESIGN: A randomized-controlled trial. SETTING: Special Rehabilitation Services in Taboão da Serra. PARTICIPANTS: Patients with bilateral knee osteoarthritis. INTERVENTION: Participants were randomly allocated to Group I (diadynamic currents and exercises; n = 30, 60 knees) or Group II (exercises alone; n = 30, 60 knees) and were treated three times a week for 8 weeks. MAIN OUTCOME MEASURES: The primary outcome measures were change in knee pain evaluated by visual analog scale and disability Index Score (Lequesne). Secondary outcomes included change in mobility (Timed Up and Go test), range of motion (goniometer), muscle strength (dynamometer), a composite score for pain and disability (Western Ontario and McMaster Universities Osteoarthritis questionnaire), and a drug diary to measure consumption of rescue pain medication (paracetamol). All measurements were collected at baseline, 8 weeks, and 6 months from baseline (follow-up). RESULTS: There were 60 participants with a mean (SD) age of 63.40 (8.20) years. Between-group differences in the follow-up (8 weeks and 6 months) were observed for pain at rest, pain during activities of daily living and disability. There was improvement in Group I that was maintained for the three variables 6 months after treatment. Mean difference for pain at rest was -3.08 points (95% confidence interval -4.13; -2.02), p < 0.01 with an effect size of 1.4; mean difference for pain during activities of daily living was -2.40 points (95% confidence interval -3.34; -1.45), p < 0.01 with an effect size of 1.24; and mean difference for disability was -4.08 points (95% confidence interval -5.89; -2.26), p < 0.01 with an effect size of 1.04. CONCLUSION: Patients with symptomatic knee osteoarthritis receiving 8 weeks of treatment with diadynamic currents as an adjunct to a program of exercises had significantly greater improvements in pain and disability than those receiving exercises alone. Beneficial effects were sustained for 6 months.

Highly Diluted Glyphosate Mitigates Its Effects on Artemia salina: Physicochemical Implications.

Glyphosate is an herbicide widely used in agriculture but can present chronic toxicity in low concentrations. Artemia salina is a common bio-indicator of ecotoxicity; it was used herein as a model to evaluate the effect of highly diluted-succussed glyphosate (potentized glyphosate) in glyphosate-based herbicide (GBH) exposed living systems. Artemia salina cysts were kept in artificial seawater with 0.02% glyphosate (corresponding to 10% lethal concentration or LC10) under constant oxygenation, luminosity, and controlled temperature, to promote hatching in 48 h. Cysts were treated with 1% (v/v) potentized glyphosate in different dilution levels (Gly 6 cH, 30 cH, 200 cH) prepared the day before according to homeopathic techniques, using GBH from the same batch. Controls were unchallenged cysts, and cysts treated with succussed water or potentized vehicle. After 48 h, the number of born nauplii per 100 µL, nauplii vitality, and morphology were evaluated. The remaining seawater was used for physicochemical analyses using solvatochromic dyes. In a second set of experiments, Gly 6 cH treated cysts were observed under different degrees of salinity (50 to 100% seawater) and GBH concentrations (zero to LC 50); hatching and nauplii activity were recorded and analyzed using the ImageJ 1.52, plug-in Trackmate. The treatments were performed blind, and the codes were revealed after statistical analysis. Gly 6 cH increased nauplii vitality (p = 0.01) and improved the healthy/defective nauplii ratio (p = 0.005) but delayed hatching (p = 0.02). Overall, these results suggest Gly 6cH treatment promotes the emergence of the more GBH-resistant phenotype in the nauplii population. Also, Gly 6cH delays hatching, another useful survival mechanism in the presence of stress. Hatching arrest was most marked in 80% seawater when exposed to glyphosate at LC10. Water samples treated with Gly 6 cH showed specific interactions with solvatochromic dyes, mainly Coumarin 7, such that it appears to be a potential physicochemical marker for Gly 6 cH. In short, Gly 6 cH treatment appears to protect the Artemia salina population exposed to GBH at low concentrations.

Pilot project. Resveratrol intake by physical active and sedentary older adult women and blood pressure.

OBJECTIVE: Although the use of resveratrol is widespread, there is a lack of studies concerning its use in older adults who practice regular physical activity. The present study aimed to evaluate how resveratrol influenced anthropometric parameters, heart measures, blood analytes in women participants, aged 60 to 80 years old, distributed in four groups consisting of those who regularly exercise or not, and those who were under resveratrol treatment or not. The older adult women exercised in a Community Center. METHODS: A prospective, comparative clinical pilot study was carried out based on convenience non-probability sampling. Resveratrol 300 mg/daily/60 days was orally-administered to 43 participants that were regularly accompanied and checked for anthropometrical parameters, heart measures, blood analytes, leukogram, and plaquetogram indices. RESULTS: The effect of resveratrol and exercise was not significant in most of the parameters evaluated in the study. Nonetheless, both systolic (p = 0.0120) and diastolic (p = 0.0241) blood pressures (BP) were elevated in sedentary older adult women in comparison with older women that regularly exercised. However, they remained within the normal BP range considered for the age. Mean corpuscular volume (MCH; p = 0.0198) and red blood cell distribution width (RDW; p < 0.0001) were improved in the groups receiving resveratrol and exercises compared to the sedentary group receiving resveratrol. The sedentary group receiving resveratrol showed diminished segmented cells (p = 0.0459), basophils (p < 0.0001), and lymphocytes (p < 0.0001). CONCLUSIONS: Present findings showed that resveratrol consumption for sedentary older women could lead to dysregulated blood pressure. Although resveratrol consumption is indicated to treat inflammation, its use must be discussed with a health professional and not be inadvertently self-administered due to significant alterations in blood pressure in older adult women, primarily if not related to exercise practice.

Environmental parameters modulate lead chloride toxicity on Artemia salina

Different environmental conditions can influence the effects of toxic agents on living beings. Recently, a series of experiments performed in Artemia salina submitted to different kinds of intoxication have shown that both, isotherapic and succussed watercan change Artemia salina ́s bio resilience at different levels. Moreover, it seems to vary according to the circalunar cycle. Objective:To verify if circalunar phases and water agitation can modify the toxicity of lead chloride on Artemia salina in vitro. Methodology:Artemia salina cysts were exposed to seawater containing 0.04% of lead chloride (equal to EC10 or 10% effective concentration, previously determined in a pilot study) in 96-well culture plates. Thirty-six experimental repetitions were performed in four series to observe the possible effects of adding stirred water, the so-called succussed water, and the moon phases. The hatched cysts were recorded after 48 hours using a digital microscope (1000x magnification) to identify the hatching percentage and the viability and mobility of the born nauplii. Results:The exposition of cysts to PbCl2 led to an increase in the hatching rate, and it was more evident during the full moon (p = 0.00014) The addition of succussed water into the seawater medium reduced this effect to the baseline levels. An increase in mobility was seen in nauplii born from exposed cysts during the full moon (p = 0.00077), but this effect was not affected by the treatment with succussed water. Discussion:Although the effects of lead chloride EC10 on the increase of nauplii hatching were expected, two environmental variables changed the sensitivity of cysts to this harmful stimulus. The circalunar cycle varied the hatching rate according to the moon phase, even in laboratory conditions, and the addition of succussed water into the medium reduced the hatching rate, even with different intensities according to the moon phase. The organization of nano and microbubbles generated after the succussion of water could be related to this protective effect and can explain, at least partially the effects of high diluted preparations on this biological context. Conclusion:Environmental factors, such as the circalunar cycle and products of water agitation, can modulate the adaptative control of hatching in Artemia salina exposed to lead chloride at EC10.

Mercury chloride toxicity attenuation of the Brine Shrimp Artemia Salinaafter treatment with Mercurius corrosivusas isotherapic

Isotherapics preparedfromtoxic substances have been described as attenuation factors for heavy metal intoxicationin aquatic animals. Herein, Artemia salinaand mercury chloride were usedas a model to identify treatment-related bioresilience. The aim was to describe the effects of Mercurius corrosivus(MC) in different potencies on Artemia salinacyst hatching and on mercury bioavailability. Artemia salinacysts were exposed to 5.0 µg/mL of mercury chloride during the hatching phase. MC6cH, 30cH, and 200cHwere prepared and poured into artificial seawater. Different controls were used (nonchallenged cysts and challenged cysts treated with water, succussed water, and Ethilicum 1cH). Four series of nine experiments were performed for4 weeks to evaluate the percentage of cyst hatchingconsidering all moon phases. Soluble total mercury (THg) levels and precipitated mercury content were also evaluated. Solvatochromic dyes were used to check for eventual physicochemical markers of MCbiological activity. Two-way analysis of variance (ANOVA) with mixed modelswas used for evaluating the effect of different treatments andthe simultaneous influence of the moon phases on the cystshatching rate, at both observation times (24 and 48 hours).When necessary, outliers were removed, using the Tukeycriterion.Thelevel of significance αwas set at 5%. Significant delay (p<0.0001) in cyst hatching was observed after treatment with MC30cH, compared with the controls. An increase inTHg concentration in seawater (p<0.0018) and of chlorine/oxygen ratio (p<0.0001) in suspended micro-aggregateswas also seen, with possiblerelation with mercury bioavailability. Specific interaction of MC30cH with the solvatochromic dye ET33 (p<0.0017) was found. The other observed potencies of Mercurius corrosivus6 and 200 cH were not significant in relation to the observed groups.The results werepostulated as being protective effects of MC30cH on Artemia salina, by improving its bioresilience.

Homeopathy for COVID-19 Prevention: Report of an Intervention at a Brazilian Service Sector Company.

BACKGROUND: COVID-19 quickly became a serious public health problem worldwide, with serious economic and social repercussions. Homeopaths around the world have been studying to find a genus epidemicus (GE) medicine that might help in the prevention and treatment of this disease. OBJECTIVE: To compare the incidence of COVID-19 between employees who received or did not receive a homeopathic GE medicine for disease prevention. METHODS: Retrospective cohort analysis. The study population comprised all employees of a service sector company in São Paulo, Brazil, and followed up by the corporate Occupational Health department. Intervention consisted of administering Arsenicum album 30cH in a one-weekly dose. Primary outcome was incidence of COVID-19 during 3-months' follow-up (April to July, 2020). RESULTS: We analyzed 1,642 of 1,703 employees without previous diagnosis of COVID-19 at onset of the study period: 53.34% of employees were referred to telework at home and did not receive intervention (Group 1, G1); 24.66% remained working on-premises in the state of São Paulo and received the intervention (Group 2, G2); 21.98% remained working on company premises in other states and did not receive intervention (Group 3, G3). Incidence rate of COVID-19 was respectively 13.35%, 0.74%, and 67.87% (p < 0.001). The odds ratio of being infected in (1) G3 versus G1 was 13.70 (95% confidence interval [CI], 10.21 to 18.39), (2) G3 versus G2 was 283.02 (95% CI, 88.98 to 900.18), and (3) G1 versus G2 was 20.66 (95% CI, 6.53 to 65.39). LIMITATIONS: The present is a retrospective analysis of a real-world experience. We could not ensure direct observed treatment, and neither could we control adherence to general prevention measures outside company premises. CONCLUSION: The incidence of COVID-19 was significantly lower amongst on-premises employees who received the GE medication in comparison to workers who did not receive the intervention (those either at other company premises or teleworking at home).

Bioresilience to Mercury Chloride of the Brine Shrimp Artemia Salina after Treatment with Homeopathic Mercurius Corrosivus.

INTRODUCTION: Finding solutions to mitigate the impact of pollution on living systems is a matter of great interest. Homeopathic preparations of toxic substances have been described in the literature as attenuation factors for intoxication. Herein, an experimental study using Artemia salina and mercury chloride was developed as a model to identify aspects related to bioresilience. AIMS: The aim of the study was to describe the effects of homeopathic Mercurius corrosivus (MC) on Artemia salina cysts hatching and on mercury bioavailability. METHODS: Artemia salina cysts were exposed to 5.0 µg/mL of mercury chloride during the hatching phase. MC potencies (6cH, 30cH, and 200cH) were prepared in sterile purified water and poured into artificial sea water. Different controls were used (non-challenged cysts and challenged cysts treated with water, succussed water, and Ethilicum 1cH). Four series of nine experiments were performed to evaluate the percentage of cyst hatching. Soluble total mercury (THg) levels and precipitated mercury content were also evaluated. Solvatochromic dyes were used to check for eventual physicochemical markers of MC biological activity. RESULTS: Significant delay (p < 0.0001) in cyst hatching was observed only after treatment with MC 30cH, compared with controls. This result was associated with an increase of THg concentration in water (p = 0.0018) and of chlorine/oxygen ratio (p < 0.0001) in suspended micraggregates, suggesting changes in mercury bioavailability. A specific interaction of MC 30cH with the solvatochromic dye ET33 (p = 0.0017) was found. CONCLUSION: Changes in hatching rate and possible changes in Hg bioavailability are postulated as protective effects of MC 30cH on Artemia salina, by improving its natural bioresilience processes.

Contributions of newborn temperature and height to Apgar index in parturient users of multiple drugs of abuse during pregnancy / Contribuição da temperatura e altura no index Apgar de recém-nascidos de parturientes usuários de múltiplas drogas de abuso durante a gestação

Objective ­ To relate anthropometric data of newborns of drug-using mothers to their children's Apgar score at birth to allow rapid intervention, if necessary. Although the Apgar has become a standard routine in assessing newborns' conditions, other variables could contribute to this index to allow rapid interventions immediately after the delivery of the newborn. Method ­ A group of mother's users of tobacco, alcohol, marijuana and/ or crack-cocaine drugs during pregnancy were selected and scores were given, taking into account the type of drug, number of drugs and their associations. At birth, the anthropometric data and the Apgar index at 1 and 5 min. were evaluated in the newborns. Statistical correlation between maternal score of dependence, the anthropometric newborn data and the Apgar index were performed. Results ­ A high correlation between the Apgar index at 1 min., the maternal score of drug dependence, the newborn's height and temperature was obtained. The coefficient of determination adjusted by Stein's equation of predictors of these variables with confidence intervals of 95% were: 1) height- 36%, 2) temperature ­ 25,7%; 3) maternal dependence -51.2%. Conclusions ­ At delivery, the height and temperature associated to the Apgar index of a newborn from mothers addicted to drugs can predict more intense care soon after birth, avoiding neonatal losses

Objetivo ­ Relacionar dados antropométricos de recém-nascidos de mães usuárias de drogas de abuso e o índice Apgar de seus filhos ao nascimento para permitir rápida intervenção, se necessária. Embora o índice Apgar tenha se tornado um padrão de rotina na avaliação das condições dos recém-nascidos, outras variáveis podem contribuir para esse índice para permitir intervenções rápidas imediatamente após o parto. Métodos ­ Um grupo de mães usuárias de tabaco, álcool, maconha e / ou cocaína crack durante a gravidez foi selecionado e pontuações foram dadas, levando em consideração o tipo de droga, número de drogas e suas associações. Ao nascimento, os dados antropométricos e o índice de Apgar aos 1 e 5 min. foram avaliados nos recém-nascidos. Foi realizada correlação estatística entre o escore materno de dependência, os dados antropométricos do recém-nascido e o índice de Apgar. Resultados ­ Foi observada alta correlação entre o índice de Apgar em 1 minuto, o escore materno da dependência de drogas, a altura e a temperatura do recém-nascido. O coeficiente de determinação ajustado pela equação de Stein de preditores dessas variáveis com intervalos de confiança de 95% foram: 1) altura ­ 36%, 2) temperatura ­ 25,7%; 3) dependência materna ­ 51,2%. Conclusões ­ No momento do parto, a altura e a temperatura associadas ao índice de Apgar de um recém-nascido de mães viciadas em drogas podem predizer cuidados mais intensos logo após o nascimento, evitando perdas neonatais.

B-1 cells upregulate CD8 T lymphocytes and increase proinflammatory cytokines serum levels in oral encephalitozoonosis.

Microsporidia are intracellular pathogens that cause severe disease in immunocompromised humans and animals. We recently demonstrated that XID mice are more susceptible to Encephalitozoon cuniculi infection by intraperitoneal route, evidencing the role of B-1 cells in resistance against infection. The present study investigated the resistance and susceptibility against E. cuniculi oral infection, including the role of B-1 cells. BALB/c and BALB/c XID (B-1 cells deficient) mice were orally infected with E. cuniculi spores. No clinical symptoms were observed in infected animals; histopathology showed lymphoplasmocytic enteritis with degeneration of the apexes of the villi in all infected groups. Higher parasite burden was observed in infected BALB/c XID mice. In the spleen and peritoneum, all infected mice showed a decrease of lymphocytes, including CD8+ T cells, mostly in infected BALB/c XID mice. Adoptive transfer of B-1 cells (XID + B-1) was associated with a lower parasite burden. Pro-inflammatory cytokines (IFN-γ, TNF-α and IL-6) increased mostly in infected XID + B1 mice. Together, the present results showed that BALB/c XID mice infected by the oral route were more susceptible to encephalitozoonosis than BALB/c mice, demonstrating the B-1 cells importance in the control of the immune response against oral E. cuniculi infection.

B-1 cells upregulate CD8 T lymphocytes and increase proinflammatory cytokines serum levels in oral encephalitozoonosis

Microsporidia are intracellular pathogens that cause severe disease in immunocompromised humans and animals. We recently demonstrated that XID mice are more susceptible to Encephalitozoon cuniculi infection by intraperitoneal route, evidencing the role of B-1 cells in resistance against infection. The present study investigated the resistance and susceptibility against E. cuniculi oral infection, including the role of B-1 cells. BALB/c and BALB/c XID (B-1 cells deficient) mice were orally infected with E. cuniculi spores. No clinical symptoms were observed in infected animals; histopathology showed lymphoplasmocytic enteritis with degeneration of the apexes of the villi in all infected groups. Higher parasite burden was observed in infected BALB/c XID mice. In the spleen and peritoneum, all infected mice showed a decrease of lymphocytes, including CD8(+) T cells, mostly in infected BALB/c XID mice. Adoptive transfer of B-1 cells (XID thorn B-1) was associated with a lower parasite burden. Pro-inflammatory cytokines (IFN-gamma, TNF-alpha and IL-6) increased mostly in infected XID + B1 mice. Together, the present results showed that BALB/c XID mice infected by the oral route were more susceptible to encephalitozoonosis than BALB/c mice, demonstrating the B-1 cells importance in the control of the immune response against oral E. cuniculi infection.

B-1 cells upregulate CD8 T lymphocytes and increase proinflammatory cytokines serum levels in oral encephalitozoonosis

Microsporidia are intracellular pathogens that cause severe disease in immunocompromised humans and animals. We recently demonstrated that XID mice are more susceptible to Encephalitozoon cuniculi infection by intraperitoneal route, evidencing the role of B-1 cells in resistance against infection. The present study investigated the resistance and susceptibility against E. cuniculi oral infection, including the role of B-1 cells. BALB/c and BALB/c XID (B-1 cells deficient) mice were orally infected with E. cuniculi spores. No clinical symptoms were observed in infected animals; histopathology showed lymphoplasmocytic enteritis with degeneration of the apexes of the villi in all infected groups. Higher parasite burden was observed in infected BALB/c XID mice. In the spleen and peritoneum, all infected mice showed a decrease of lymphocytes, including CD8(+) T cells, mostly in infected BALB/c XID mice. Adoptive transfer of B-1 cells (XID thorn B-1) was associated with a lower parasite burden. Pro-inflammatory cytokines (IFN-gamma, TNF-alpha and IL-6) increased mostly in infected XID + B1 mice. Together, the present results showed that BALB/c XID mice infected by the oral route were more susceptible to encephalitozoonosis than BALB/c mice, demonstrating the B-1 cells importance in the control of the immune response against oral E. cuniculi infection.

Diabetes mellitus increases the susceptibility to encephalitozoonosis in mice.

Microsporidiosis are diseases caused by opportunistic intracellular fungi in immunosuppressed individuals, as well as in transplanted patients, the elderly and children, among others. Diabetes mellitus (DM) is a metabolic disease characterized by hyperglycemia and decreased T cell response, neutrophil function, humoral immunity failure, increasing the susceptibility to infections. Here, we investigated the susceptibility of streptozotocin (STZ)-induced type I diabetic and/or immunosuppressed mice to encephalitozoonosis by Encephalitozoon cuniculi. Microscopically, granulomatous hepatitis, interstitial pneumonia and pielonephritis were observed in all infected groups. STZ treatment induced an immunossupressor effect in the populations of B (B-1 and B2) and CD4+ T lymphocytes. Moreover, infection decreased CD4+ and CD8+ T lymphocytes and macrophages of DM mice. Furthermore, infection induced a significant increase of IL-6 and TNF-α cytokine serum levels in DM mice. IFN-γ, the most important cytokine for the resolution of encephalitozoonosis, increased only in infected mice. In addition to the decreased immune response, DM mice were more susceptible to encephalitozoonosis, associated with increased fungal burden, and symptoms. Additionally, cyclophosphamide immunosuppression in DM mice further increased the susceptibility to encephalitozoonosis. Thus, microsporidiosis should be considered in the differential diagnosis of comorbidities in diabetics.

Interface between breast cancer cells and the tumor microenvironment using platelet-rich plasma to promote tumor angiogenesis - influence of platelets and fibrin bundles on the behavior of breast tumor cells.

Cancer progression is associated with an evolving tissue interface of direct epithelial-tumor microenvironment interactions. In biopsies of human breast tumors, extensive alterations in molecular pathways are correlated with cancer staging on both sides of the tumor-stroma interface. These interactions provide a pivotal paracrine signaling to induce malignant phenotype transition, the epithelial-mesenchymal transition (EMT). We explored how the direct contact between platelets-fibrin bundles primes metastasis using platelet-rich plasma (PRP) as a source of growth factors and mimics the provisional fibrin matrix between actively growing breast cancer cells and the tumor stroma. We have demonstrated PRP functions, modulating cell proliferation that is tumor-subtype and cancer cell-type-specific. Epithelial and stromal primary cells were prepared from breast cancer biopsies from 21 women with different cancer subtypes. Cells supplemented with PRP were immunoblotted with anti-phospho and total Src-Tyr-416, FAK-Try-925, E-cadherin, N-cadherin, TGF-ß, Smad2, and Snail monoclonal antibodies. Breast tumor cells from luminal B and HER2 subtypes showed the most malignant profiles and the expression of thrombin and other classes of proteases at levels that were detectable through FRET peptide libraries. The angiogenesis process was investigated in the interface obtained between platelet-fibrin-breast tumor cells co-cultured with HUVEC cells. Luminal B and HER2 cells showed robust endothelial cell capillary-like tubes ex vivo. The studied interface contributes to the attachment of endothelial cells, provides a source of growth factors, and is a solid substrate. Thus, replacement of FBS supplementation with PRP supplementation represents an efficient and simple approach for mimicking the real multifactorial tumor microenvironment.

Diabetes mellitus increases the susceptibility to encephalitozoonosis in mice

Microsporidiosis are diseases caused by opportunistic intracellular fungi in immunosuppressed individuals, as well as in transplanted patients, the elderly and children, among others. Diabetes mellitus (DM) is a metabolic disease characterized by hyperglycemia and decreased T cell response, neutrophil function, humoral immunity failure, increasing the susceptibility to infections. Here, we investigated the susceptibility of streptozotocin (STZ)-induced type I diabetic and/or immunosuppressed mice to encephalitozoonosis by Encephalitozoon cuniculi. Microscopically, granulomatous hepatitis, interstitial pneumonia and pielonephritis were observed in all infected groups. STZ treatment induced an immunossupressor effect in the populations of B (B-1 and B2) and CD4+ T lymphocytes. Moreover, infection decreased CD4+ and CD8+ T lymphocytes and macrophages of DM mice. Furthermore, infection induced a significant increase of IL-6 and TNF-a cytokine serum levels in DM mice. IFN-gama, the most important cytokine for the resolution of encephalitozoonosis, increased only in infected mice. In addition to the decreased immune response, DM mice were more susceptible to encephalitozoonosis, associated with increased fungal burden, and symptoms. Additionally, cyclophosphamide immunosuppression in DM mice further increased the susceptibility to encephalitozoonosis. Thus, microsporidiosis should be considered in the differential diagnosis of comorbidities in diabetics

Cathepsin K induces platelet dysfunction and affects cell signaling in breast cancer - molecularly distinct behavior of cathepsin K in breast cancer.

BACKGROUND: Breast cancer comprises clinically and molecularly distinct tumor subgroups that differ in cell histology and biology and show divergent clinical phenotypes that impede phase III trials, such as those utilizing cathepsin K inhibitors. Here we correlate the epithelial-mesenchymal-like transition breast cancer cells and cathepsin K secretion with activation and aggregation of platelets. Cathepsin K is up-regulated in cancer cells that proteolyze extracellular matrix and contributes to invasiveness. Although proteolytically activated receptors (PARs) are activated by proteases, the direct interaction of cysteine cathepsins with PARs is poorly understood. In human platelets, PAR-1 and -4 are highly expressed, but PAR-3 shows low expression and unclear functions. METHODS: Platelet aggregation was monitored by measuring changes in turbidity. Platelets were immunoblotted with anti-phospho and total p38, Src-Tyr-416, FAK-Tyr-397, and TGFß monoclonal antibody. Activation was measured in a flow cytometer and calcium mobilization in a confocal microscope. Mammary epithelial cells were prepared from the primary breast cancer samples of 15 women with Luminal-B subtype to produce primary cells. RESULTS: We demonstrate that platelets are aggregated by cathepsin K in a dose-dependent manner, but not by other cysteine cathepsins. PARs-3 and -4 were confirmed as the cathepsin K target by immunodetection and specific antagonists using a fibroblast cell line derived from PARs deficient mice. Moreover, through co-culture experiments, we show that platelets activated by cathepsin K mediated the up-regulation of SHH, PTHrP, OPN, and TGFß in epithelial-mesenchymal-like cells from patients with Luminal B breast cancer. CONCLUSIONS: Cathepsin K induces platelet dysfunction and affects signaling in breast cancer cells.

α- L-iduronidase gene-based therapy using the phiC31 system to treat mucopolysaccharidose type I mice.

BACKGROUND: Mucopolysaccharidose type I (MPSI) is a lysosomal monogenic disease caused by mutations in the gene for α- L-iduronidase (IDUA). MPSI patients need a constant supply of IDUA to alleviate progression of the disease. IDUA gene transfer using integrative vectors might provide a definitive solution and support advancement to clinical trials, although studies have not yet been satisfactory. To achieve a stable IDUA gene expression in vivo, phiC31 was tested in the present study. METHODS: Several plasmid vectors were constructed and IDUA-/- mice were treated with cyclophosphamide and transfected with these vectors hydrodynamically via tail veins. IDUA expression was monitored over time. Treated and nontreated mice underwent an open-field test at age 8 months, and IDUA activity and glycosaminoglycan (GAG) content of tissues were evaluated. RESULTS: High levels of IDUA activity were detected initially (>1000 U/ml), although these levels decayed over time. The reinjection of vectors produced a similar profile of IDUA decay. Three out of six treated mice had IDUA activity in the livers, and also showed lower GAG content, reduced lysosomes and better locomotion. To investigate unsustained IDUA production, wild-type mice were submitted to the same gene therapy procedure, which generated a similar profile of IDUA decay. Anti-IDUA antibody was detected in the sera of these animals. In addition, we also found three methylated sites in the cytomegalovirus promoter region. CONCLUSIONS: phiC31-mediated gene therapy resulted in an important improvement in IDUA-/- mice, including locomotion, although the obstacles that need to be overcome to enable long-term gene therapy for MPSI are also noted.

Mesenchymal stem cells do not prevent antibody responses against human α-L-iduronidase when used to treat mucopolysaccharidosis type I.

Mucopolysaccharidosis type I (MPSI) is an autosomal recessive disease that leads to systemic lysosomal storage, which is caused by the absence of α-L-iduronidase (IDUA). Enzyme replacement therapy is recognized as the best therapeutic option for MPSI; however, high titers of anti-IDUA antibody have frequently been observed. Due to the immunosuppressant properties of MSC, we hypothesized that MSC modified with the IDUA gene would be able to produce IDUA for a long period of time. Sleeping Beauty transposon vectors were used to modify MSC because these are basically less-immunogenic plasmids. For cell transplantation, 4×10(6) MSC-KO-IDUA cells (MSC from KO mice modified with IDUA) were injected into the peritoneum of KO-mice three times over intervals of more than one month. The total IDUA activities from MSC-KO-IDUA before cell transplantation were 9.6, 120 and 179 U for the first, second and third injections, respectively. Only after the second cell transplantation, more than one unit of IDUA activity was detected in the blood of 3 mice for 2 days. After the third cell transplantation, a high titer of anti-IDUA antibody was detected in all of the treated mice. Anti-IDUA antibody response was also detected in C57Bl/6 mice treated with MSC-WT-IDUA. The antibody titers were high and comparable to mice that were immunized by electroporation. MSC-transplanted mice had high levels of TNF-alpha and infiltrates in the renal glomeruli. The spreading of the transplanted MSC into the peritoneum of other organs was confirmed after injection of 111In-labeled MSC. In conclusion, the antibody response against IDUA could not be avoided by MSC. On the contrary, these cells worked as an adjuvant that favored IDUA immunization. Therefore, the humoral immunosuppressant property of MSC is questionable and indicates the danger of using MSC as a source for the production of exogenous proteins to treat monogenic diseases.