Kidney injury and cell therapy: Preclinical study

The aim of this study is to show histological and immunofluorescence analysis of renal parenchyma of agoutis affected by gentamicin-induced renal disease after the infusion of bone marrow mononuclear cells (BMMC) stained with Hoechst (R). Nine agouti's males were divided into three groups: Test group (TG): renal disease by gentamicin induced (n = 3), cell therapy group (CTG): renal disease by gentamicin induced and BMMC infusion (n = 3), and control group (CG): nonrenal disease and BMMC infusion (n = 3). TG and CTG were submitted to the protocol of renal disease induction using weekly application of gentamicin sulfate for 4 months. CG and CTG received a 1 X 108 BMMC stained with Hoechst and were euthanized for kidney examination 21 days after BMMC injection and samples were collected for histology and immunofluorescence analysis. Histological analysis demonstrated typical interstitial lesions in kidney similarly to human disease, as tubular necrosis, glomerular destruction, atrophy tubular, fibrotic areas, and collagen deposition. We conclude that histological analysis suggest a positive application of agouti's as a model for a gentamicin inducing of kidney disease, beyond the immunofluorescence analysis suggest a significant migration of BMMC to sites of renal injury in CTG. Microsc. Res. Tech., 2012. (c) 2011 Wiley Periodicals, Inc.

Celecoxib treatment does not alter recruitment and activation of osteoclasts in the initial phase of experimental tooth movement

In a previous study, we reported that the short-term treatment with celecoxib, a nonsteroidal anti-inflammatory drug (NSAID) attenuates the activation of brain structures related to nociception and does not interfere with orthodontic incisor separation in rats. The conclusion was that celecoxib could possibly be prescribed for pain in orthodontic patients. However, we did not analyze the effects of this drug in periodontium. The aim of this follow-up study was to analyze effects of celecoxib treatment on recruitment and activation of osteoclasts and alveolar bone resorption after inserting an activated orthodontic appliance between the incisors in our rat model. Twenty rats (400420 g) were pretreated through oral gavage with celecoxib (50 mg/kg) or vehicle (carboxymethylcellulose 0.4%). After 30 min, they received an activated (30 g) orthodontic appliance, set not to cause any palate disjunction. In sham animals, the appliance was immediately removed after introduction. All animals received ground food and, every 12 h, celecoxib or vehicle. After 48 h, they were anesthetized and transcardiacally perfused through the aorta with 4% formaldehyde. Subsequently, maxillae were removed, post-fixed and processed for histomorphometry or immunohistochemical analyses. As expected, incisor distalization induced an inflammatory response with certain histological changes, including an increase in the number of active osteoclasts at the compression side in group treated with vehicle (appliance: 32.2 +/- 2.49 vs sham: 4.8 +/- 1.79, P<0.05) and celecoxib (appliance: 31.0 +/- 1.45 vs sham: 4.6 +/- 1.82, P<0.05). The treatment with celecoxib did not modify substantially the histological alterations and the number of active osteoclasts after activation of orthodontic appliance. Moreover, we did not see any difference between the groups with respect to percentage of bone resorption area. Taken together with our previous results we conclude that short-term treatment with celecoxib can indeed be a therapeutic alternative for pain relieve during orthodontic procedures.

Bosentan, an endothelin receptor antagonist, ameliorates collagen-induced arthritis: the role of TNF-alpha in the induction of endothelin system genes

Endothelins (ETs) are involved in several inflammatory events. The present study investigated the efficacy of bosentan, a dual ETA/ETB receptor antagonist, in collagen-induced arthritis (CIA) in mice. CIA was induced in DBA/1J mice. Arthritic mice were treated with bosentan (100 mg/kg) once a day, starting from the day when arthritis was clinically detectable. CIA progression was assessed by measurements of visual clinical score, paw swelling and hypernociception. Histological changes, neutrophil infiltration and pro-inflammatory cytokines were evaluated in the joints. Gene expression in the lymph nodes of arthritic mice was evaluated by microarray technology. PreproET-1 mRNA expression in the lymph nodes of mice and in peripheral blood mononuclear cells (PBMCs) was evaluated by real-time PCR. The differences were evaluated by one-way ANOVA or Student's t test. Oral treatment with bosentan markedly ameliorated the clinical aspects of CIA (visual clinical score, paw swelling and hyperalgesia). Bosentan treatment also reduced joint damage, leukocyte infiltration and pro-inflammatory cytokine levels (IL-1 beta, TNF alpha and IL-17) in the joint tissues. Changes in gene expression in the lymph nodes of arthritic mice returned to the levels of the control mice after bosentan treatment. PreproET mRNA expression increased in PBMCs from rheumatoid arthritis (RA) patients but returned to basal level in PBMCs from patients under anti-TNF therapy. In-vitro treatment of PBMCs with TNF alpha upregulated ET system genes. These findings indicate that ET receptor antagonists, such as bosentan, might be useful in controlling RA. Moreover, it seems that ET mediation of arthritis is triggered by TNF alpha.

Infrared (810-nm) low-level laser therapy on rat experimental knee inflammation

Arthritis of the knee is the most common type of joint inflammatory disorder and it is associated with pain and inflammation of the joint capsule. Few studies address the effects of the 810-nm laser in such conditions. Here we investigated the effects of low-level laser therapy (LLLT; infrared, 810-nm) in experimentally induced rat knee inflammation. Thirty male Wistar rats (230-250 g) were anesthetized and injected with carrageenan by an intra-articular route. After 6 and 12 h, all animals were killed by CO(2) inhalation and the articular cavity was washed for cellular and biochemical analysis. Articular tissue was carefully removed for real-time PCR analysis in order to evaluate COX-1 and COX-2 expression. LLLT was able to significantly inhibit the total number of leukocytes, as well as the myeloperoxidase activity with 1, 3, and 6 J (Joules) of energy. This result was corroborated by cell counting showing the reduction of polymorphonuclear cells at the inflammatory site. Vascular extravasation was significantly inhibited at the higher dose of energy of 10 J. Both COX-1 and 2 gene expression were significantly enhanced by laser irradiation while PGE(2) production was inhibited. Low-level laser therapy operating at 810 nm markedly reduced inflammatory signs of inflammation but increased COX-1 and 2 gene expression. Further studies are necessary to investigate the possible production of antiinflammatory mediators by COX enzymes induced by laser irradiation in knee inflammation.

Dental Pulp Vascular Permeability Changes Induced by Dental Bleaching

Aiming to compare the effect of different light sources for dental bleaching on vascular permeability of dental pulps, forty-eight incisors were used. The bleaching agent (35% hydrogen peroxide) was activated by halogen light; LED (Light Emitting Diode) or LED, followed by laser phototherapy (LPT) (lambda = 780 nm; 3 J/cm(2)). After the bleaching procedures, the animals received an intra-arterial dye injection and one hour later were sacrificed. The teeth were diaphanized and photographed. The amount of blue stain content of each dental pulp was quantified using a computer imaging program. The data was statistically compared (p <= 0.05). The results showed a significant higher (p <= 0.01) dye content in the groups bleached with halogen light, compared with the control, LED and LED plus LPT groups. Thus, tooth bleaching activated by LED or LED plus LPT induces lesser resulted in increased vascular permeability than halogen light.

Differential Gene Expression Profiles May Differentiate Responder and Nonresponder Patients with Rheumatoid Arthritis for Methotrexate (MTX) Monotherapy and MTX plus Tumor Necrosis Factor Inhibitor Combined Therapy

Objective. We aimed to evaluate whether the differential gene expression profiles of patients with rheumatoid arthritis (RA) could distinguish responders from nonresponders to methotrexate (MTX) and, in the case of MTX nonresponders, responsiveness to MTX plus anti-tumor necrosis factor-alpha (anti-TNF) combined therapy. Methods. We evaluated 25 patients with RA taking MTX 15-20 mg/week as a monotherapy (8 responders and 17 nonresponders). All MTX nonresponders received intliximab and were reassessed after 20 weeks to evaluate their anti-TNF responsiveness using the European League Against Rheumatism response criteria. A differential gene expression analysis from peripheral blood mononuclear cells was performed in terms of hierarchical gene clustering, and an evaluation of differentially expressed genes was performed using the significance analysis of microarrays program. Results. Hierarchical gene expression clustering discriminated MTX responders from nonresponders, and MTX plus anti-TNF responders from nonresponders. The evaluation of only highly modulated genes (fold change > 1.3 or < 0.7) yielded 5 induced (4 antiapoptotic and CCL4) and 4 repressed (4 proapoptotic) genes in MTX nonresponders compared to responders. In MTX plus anti-TNF nonresponders, the CCL4, CD83, and BCL2A1 genes were induced in relation to responders. Conclusion. Study of the gene expression profiles of RA peripheral blood cells permitted differentiation of responders from nonresponders to MTX and anti-TNF. Several candidate genes in MTX non-responders (CCL4, HTRA2, PRKCD, BCL2A1, CAV1, TNIP1 CASP8AP2, MXD1, and BTG2) and 3 genes in MTX plus anti-TNF nonresponders (CCL4, CD83, and BCL2A1) were identified for further study. (First Release July 1 2012; J Rheumatol 2012;39:1524-32; doi:10.3899/jrheum.120092)

Fluorescence spectroscopy as a tool to detect and evaluate glucocorticoid-induced skin atrophy

Topical glucocorticoid (GC) therapy has been successfully used in the treatment of several common cutaneous diseases in clinical practice for a long time, and skin atrophy is one of the most typical cutaneous side effects of this therapy. The aim of this study was to evaluate the potential of noninvasive fluorescence spectroscopy (FS) technique in the detection and classification of GC-induced skin atrophy. A total of 20 male Wistar rats were used in the experimental protocol under controlled environmental conditions and with free access to food. One group received topical application of clobetasol propionate 0.05% for 14 days to induce cutaneous atrophy (atrophic group) and the other (control) group received only vehicle application following the same protocol and schedule. Histological analyses and FS measurements with laser excitation at both 532 nm and 408 nm were obtained on days 1 and 15. The FS results were classified as "normal" or "atrophic" according by histological analysis. Fluorescence spectra obtained with excitation at 408 nm allowed a clear distinction between the control and atrophic groups, and were more informative than the those obtained at 532 nm. Our results reveal that, if correctly applied, FS allows noninvasive evaluation of corticosteroid-induced skin atrophy, and thus represents an important step towards better monitoring of undesirable side effects of cutaneous therapy.

Antimicrobial Photodynamic Therapy for the Treatment of Teeth with Apical Periodontitis: A Histopathological Evaluation

Introduction: This study evaluated the in vivo response of apical and periapical tissues of dogs' teeth with apical periodontitis after one-session endodontic treatment with and without antimicrobial photodynamic therapy (aPDT). Methods: Sixty root canals with experimentally induced apical periodontitis were instrumented and assigned to 4 groups receiving aPDT and root canal filling (RCF) or not: group aPDT+/RCF- (n = 20): aPDT (photosensitizer phenothiazine chloride at 10 mg/mL for 3 minutes and diode laser [2 = 660 nm, 60 mW/cm(2)] for 1 minute) and RCF in the same session; group aPDT+/RCF (n = 10); group aPDT /RCF+ (n = 20), and group aPDT /RCF (n = 10). Teeth were restored, and the animals were killed after 90 days. Sections from the maxillas and mandibles were stained with hematoxylin-eosin and Mallory trichrome and examined under light microscopy. Descriptive (ie, newly formed apical mineralized tissue, periapical inflammatory infiltrate, apical periodontal ligament thickness, and mineralized tissue resorption) and quantitative (ie, periapical lesion size and number of inflammatory cells) microscopic analysis was performed. Quantitative data were analyzed by the Kruskal-Wallis and Dunn tests (alpha =.05). Results: In the aPDT-treated groups, the periapical region was moderately/severely enlarged with no inflammatory cells, moderate neoangiogenesis and fibrogenesis, and the smallest periapical lesions. Conclusions: Although apical closure by mineralized tissue deposition was not achieved, the absence of inflammatory cells, moderate neoangiogenesis, and fibrogenesis in the periapical region in the groups treated with aPDT indicate that this can be a promising adjunct therapy to cleaning and shaping procedures in teeth with apical periodontitis undergoing one-session endodontic treatment. (J Endod 2012;38:360-366)

Pathogen-Induced Proapoptotic Phenotype and High CD95 (Fas) Expression Accompany a Suboptimal CD8(+) T-Cell Response: Reversal by Adenoviral Vaccine

MHC class la-restricted CD8(+) T cells are important mediators of the adaptive immune response against infections caused by intracellular microorganisms. Whereas antigen-specific effector CD8(+) T cells can clear infection caused by intracellular pathogens, in some circumstances, the immune response is suboptimal and the microorganisms survive, causing host death or chronic infection. Here, we explored the cellular and molecular mechanisms that could explain why CD8(+) T-cell-mediated immunity during infection with the human protozoan parasite Trypanosoma cruzi is not optimal. For that purpose, we compared the CD8(+) T-cell mediated immune responses in mice infected with T. cruzi or vaccinated with a recombinant adenovirus expressing an immunodominant parasite antigen. Several functional and phenotypic characteristics of specific CD8(+) T cells overlapped. Among few exceptions was an accelerated expansion of the immune response in adenoviral vaccinated mice when compared to infected ones. Also, there was an upregulated expression of the apoptotic-signaling receptor CD95 on the surface of specific T cells from infected mice, which was not observed in the case of adenoviral-vaccinated mice. Most importantly, adenoviral vaccine provided at the time of infection significantly reduced the upregulation of CD95 expression and the proapoptotic phenotype of pathogen-specific CD8(+) cells expanded during infection. In parallel, infected adenovirus-vaccinated mice had a stronger CD8(+) T-cell mediated immune response and survived an otherwise lethal infection. We concluded that a suboptimal CD8(+) T-cell response is associated with an upregulation of CD95 expression and a proapoptotic phenotype. Both can be blocked by adenoviral vaccination.

Effect of Cholecalciferol as Adjunctive Therapy With Insulin on Protective Immunologic Profile and Decline of Residual beta-Cell Function in New-Onset Type 1 Diabetes Mellitus

Objective: To evaluate the effect of vitamin D-3 on cytokine levels, regulatory T cells, and residual beta-cell function decline when cholecalciferol (vitamin D-3 administered therapeutically) is given as adjunctive therapy with insulin in new-onset type 1 diabetes mellitus (T1DM). Design and Setting: An 18-month (March 10, 2006, to October 28, 2010) randomized, double-blind, placebo-controlled trial was conducted at the Diabetes Center of Sao Paulo Federal University, Sao Paulo, Brazil. Participants: Thirty-eight patients with new-onset T1DM with fasting serum C-peptide levels greater than or equal to 0.6 ng/mL were randomly assigned to receive daily oral therapy of cholecalciferol, 2000 IU, or placebo. Main Outcome Measure: Levels of proinflammatory and anti-inflammatory cytokines, chemokines, regulatory T cells, hemoglobin A(1c), and C-peptide; body mass index; and insulin daily dose. Results: Mean (SD) chemokine ligand 2 (monocyte chemoattractant protein 1) levels were significantly higher (184.6 [101.1] vs 121.4 [55.8] pg/mL) at 12 months, as well as the increase in regulatory T-cell percentage (4.55%[1.5%] vs 3.34%[1.8%]) with cholecalciferol vs placebo. The cumulative incidence of progression to undetectable (<= 0.1 ng/mL) fasting C-peptide reached 18.7% in the cholecalciferol group and 62.5% in the placebo group; stimulated C-peptide reached 6.2% in the cholecalciferol group and 37.5% in the placebo group at 18 months. Body mass index, hemoglobin A(1c) level, and insulin requirements were similar between the 2 groups. Conclusions: Cholecalciferol used as adjunctive therapy with insulin is safe and associated with a protective immunologic effect and slow decline of residual beta-cell function in patients with new-onset T1DM. Cholecalciferol may be an interesting adjuvant in T1DM prevention trials.

Low-level laser therapy (LLLT) in human progressive-intensity running: effects on exercise performance, skeletal muscle status, and oxidative stress

The aim of this work was to evaluate the effects of low-level laser therapy (LLLT) on exercise performance, oxidative stress, and muscle status in humans. A randomized double-blind placebo-controlled crossover trial was performed with 22 untrained male volunteers. LLLT (810 nm, 200 mW, 30 J in each site, 30 s of irradiation in each site) using a multi-diode cluster (with five spots - 6 J from each spot) at 12 sites of each lower limb (six in quadriceps, four in hamstrings, and two in gastrocnemius) was performed 5 min before a standardized progressive-intensity running protocol on a motor-drive treadmill until exhaustion. We analyzed exercise performance (VO(2 max), time to exhaustion, aerobic threshold and anaerobic threshold), levels of oxidative damage to lipids and proteins, the activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), and the markers of muscle damage creatine kinase (CK) and lactate dehydrogenase (LDH). Compared to placebo, active LLLT significantly increased exercise performance (VO(2 max) p = 0.01; time to exhaustion, p = 0.04) without changing the aerobic and anaerobic thresholds. LLLT also decreased post-exercise lipid (p = 0.0001) and protein (p = 0.0230) damages, as well as the activities of SOD (p = 0.0034), CK (p = 0.0001) and LDH (p = 0.0001) enzymes. LLLT application was not able to modulate CAT activity. The use of LLLT before progressive-intensity running exercise increases exercise performance, decreases exercise-induced oxidative stress and muscle damage, suggesting that the modulation of the redox system by LLLT could be related to the delay in skeletal muscle fatigue observed after the use of LLLT.

Spiritist Psychiatric Hospitals in Brazil: Integration of Conventional Psychiatric Treatment and Spiritual Complementary Therapy

In Brazil, during the XX century, dozens of Spiritist psychiatric hospitals emerged seeking to integrate conventional medical treatment with complementary spiritual therapy. This combined inpatient treatment is largely found in Brazil, where many psychiatric hospitals stem from the Spiritist movement. The present report describes the use of these spiritual practices, their operating structure, health professionals involved, modalities of care, and institutional difficulties in integrating spiritual practices with conventional treatment in six leading Brazilian Spiritist psychiatric hospitals. These hospitals combine conventional psychiatric treatment with voluntary-based spiritual approaches such as laying on of hands ("fluidotherapy"), lectures regarding spiritual and ethical issues, intercessory prayer, spirit release therapy ("disobsession") and "fraternal dialogue". The non-indoctrination and optional nature of these spiritual complementary therapies seem to increase acceptance among patients and their family members. In conclusion, the Spiritist psychiatric hospitals in Brazil have, for more than half a century, provided an integrative approach in the treatment of psychiatric disorders, associating conventional and spiritual treatments, more specifically Spiritist therapy. The lack of standardized treatment protocols and scientific studies remain a barrier to assessing the impact of this integrative approach on patients' mental health, quality of life, adherence, and perceived quality of treatment.

In vivo study of laser irradiation of fractionated drug administration based mechanism for effective photodynamic therapy in rat liver

Up-regulation of stress-activated proteins in cancer cells plays a protective role against photodynamic induced apoptosis. Post photodynamic therapy extracted normal rat liver tissue usually shows a fraction of surviving cells, the photodynamic resistant cells, residing in the necrotic region. To treat these photo-dynamic resistant cells a technique has been proposed based on fractionated drug administration of diluted photosensitizer, keeping the net concentration (5 mg/kg) constant, and subsequently varying drug light interval (DLI). Flourescence measurements were made for the presence of photosensitizer in a tissue. For qualitative analysis both histological and morphological studies were made. Although preliminary aim of this approach was not achieved but there were some interesting observation made i.e. for higher dilution of photosensitizer there was a sharp boundary between necrotic and normal portion of tissue. An increase in the absorption coefficient (alpha) from 2.7 -> 2.9 was observed as photosensitizer was diluted while the corresponding threshold dose (D (th)) persistently decreases from (0.10 -> 0.02) J/cm(2) when irradiated with a 635 nm laser fluence of 150 J/cm(2).

Free Rhodium (II) citrate and rhodium (II) citrate magnetic carriers as potential strategies for breast cancer therapy

Abstract Background Rhodium (II) citrate (Rh2(H2cit)4) has significant antitumor, cytotoxic, and cytostatic activity on Ehrlich ascite tumor. Although toxic to normal cells, its lower toxicity when compared to carboxylate analogues of rhodium (II) indicates Rh2(H2cit)4 as a promising agent for chemotherapy. Nevertheless, few studies have been performed to explore this potential. Superparamagnetic particles of iron oxide (SPIOs) represent an attractive platform as carriers in drug delivery systems (DDS) because they can present greater specificity to tumor cells than normal cells. Thus, the association between Rh2(H2cit)4 and SPIOs can represent a strategy to enhance the former's therapeutic action. In this work, we report the cytotoxicity of free rhodium (II) citrate (Rh2(H2cit)4) and rhodium (II) citrate-loaded maghemite nanoparticles or magnetoliposomes, used as drug delivery systems, on both normal and carcinoma breast cell cultures. Results Treatment with free Rh2(H2cit)4 induced cytotoxicity that was dependent on dose, time, and cell line. The IC50 values showed that this effect was more intense on breast normal cells (MCF-10A) than on breast carcinoma cells (MCF-7 and 4T1). However, the treatment with 50 μM Rh2(H2cit)4-loaded maghemite nanoparticles (Magh-Rh2(H2cit)4) and Rh2(H2cit)4-loaded magnetoliposomes (Lip-Magh-Rh2(H2cit)4) induced a higher cytotoxicity on MCF-7 and 4T1 than on MCF-10A (p < 0.05). These treatments enhanced cytotoxicity up to 4.6 times. These cytotoxic effects, induced by free Rh2(H2cit)4, were evidenced by morphological alterations such as nuclear fragmentation, membrane blebbing and phosphatidylserine exposure, reduction of actin filaments, mitochondrial condensation and an increase in number of vacuoles, suggesting that Rh2(H2cit)4 induces cell death by apoptosis. Conclusions The treatment with rhodium (II) citrate-loaded maghemite nanoparticles and magnetoliposomes induced more specific cytotoxicity on breast carcinoma cells than on breast normal cells, which is the opposite of the results observed with free Rh2(H2cit)4 treatment. Thus, magnetic nanoparticles represent an attractive platform as carriers in Rh2(H2cit)4 delivery systems, since they can act preferentially in tumor cells. Therefore, these nanopaticulate systems may be explored as a potential tool for chemotherapy drug development.

Dietary restriction abrogates antibody production induced by a DNA vaccine encoding the mycobacterial 65 kDa heat shock protein

Abstract Background Protein-calorie malnutrition (PCM) is the most common type of malnutrition. PCM leads to immunodeficiency and consequent increased susceptibility to infectious agents. In addition, responses to prophylactic vaccines depend on nutritional status. This study aims to evaluate the ability of undernourished mice to mount an immune response to a genetic vaccine (pVAXhsp65) against tuberculosis, containing the gene coding for the heat shock protein 65 from mycobacteria. Methods Young adult female BALB/c mice were fed ad libitum or with 80% of the amount of food consumed by a normal diet group. We initially characterized a mice model of dietary restriction by determining body and spleen weights, hematological parameters and histopathological changes in lymphoid organs. The ability of splenic cells to produce IFN-gamma and IL-4 upon in vitro stimulation with LPS or S. aureus and the serum titer of specific IgG1 and IgG2a anti-hsp65 antibodies after intramuscular immunization with pVAXhsp65 was then tested. Results Dietary restriction significantly decreased body and spleen weights and also the total lymphocyte count in blood. This restriction also determined a striking atrophy in lymphoid organs as spleen, thymus and lymphoid tissue associated with the small intestine. Specific antibodies were not detected in mice submitted to dietary restriction whereas the well nourished animals produced significant levels of both, IgG1 and IgG2a anti-hsp65. Conclusion 20% restriction in food intake deeply compromised humoral immunity induced by a genetic vaccine, alerting, therefore, for the relevance of the nutritional condition in vaccination programs based on these kinds of constructs.