Diagnostic accuracy of neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio, and neutrophil-lymphocyte-to-platelet ratio biomarkers in predicting bacteremia and sepsis in immunosuppressive patients with cancer: literature review.

Background: This literature review explores the role of neutrophil-to-lymphocyte ratio (NLR), platelet-to-lymphocyte ratio (PLR), and neutrophil-lymphocyte-to-platelet ratio (NLPR) biomarkers, as potential indicators for predicting bacteremia and sepsis in patients with cancer. Objective: Tracing the evolution of interest in this area since 2001, the aim of this review was to report a comprehensive overview of current knowledge and gaps, particularly in patients undergoing immunosuppression. Summary of Findings: The literature research indicates the potential of NLR, PLR, and other biomarkers in diagnosing and predicting sepsis, with some studies emphasizing their value in mortality prediction. A specific focus on bacteremia shows the effectiveness of NLR and PLR as early indicators and prognostic tools, though mostly in noncancer patient populations. While NLR and PLR are promising in general cancer patient populations, the review addresses the challenges in applying these biomarkers to patients with neutropenic and lymphopenic cancer. The NLPR could be considered a significant biomarker for inflammation and mortality risk in various medical conditions, yet its diagnostic accuracy in patients with immunosuppressed cancer is not extensively validated. Conclusion: This review offers a snapshot of the current research on biomarkers in patients with immunocompromised cancer in the sepsis and bacteremia area. More focused research on their application is necessary. This gap underscores an opportunity for future studies to enhance diagnostic and prognostic capabilities in this high-risk group.

Gastric MALT Lymphoma: A 8-Year Experience.

Gastric mucosa-associated lymphoid tissue non-Hodgkin lymphoma (gMALT NHL) is the second most common gastrointestinal lymphoma (50% of all gastric lymphomas), being closely associated with Helicobacter pylori infection, justifying that antibiotic therapy is effective in over 75% of all cases. This is a retrospective study analyzing all adult gMALT NHL cases diagnosed and treated in a single center for 8 years, focusing on demographic features, treatment outcomes, and survival analysis. Sixty patients with a median age of 61 years (53.3% female gender) were analyzed. Most of the cases had localized disease (66.7% were Lugano stage I) and had low IPI scores (median: 1). There was a high prevalence of Helicobacter pylori infection (68.3%). Nearly 97% of the cases received treatment for the disease, a median of one line; 55% of the patients treated endured complete response after first-line therapy (mostly antibiotics). Median overall survival time and median progression-free survival time were not reached. The mean follow-up time was 81.8 months (95% CI: [73.3-90.3]). Thirty-six patients (60%) achieved a 3-year follow-up time; the mortality rate was 15% at the end of the study. Age superior to 65 years and transformation into DLBCL were statistically significant negative prognostic markers for survival in this study (p = 0.006 and p = 0.033, respectively). Our study confirms that gMALT NHL is an indolent disease with long-term survival. Many patients, however, are exposed to several treatment lines along their disease course.

Gastric Diffuse Large B-Cell Lymphoma: A Single-Center 9-Year Experience.

Gastric diffuse large B cell lymphoma (DLBCL) represents the majority of all gastric lymphomas. We report a series of gastric DLBCL diagnosed and treated in a single center, between 2010 and 2018 (included). We retrospectively analyzed the population demographic features, treatment outcomes and survival. One-hundred-and-one patients were studied, 50.5% males and median age of 64 years [23-94]. Lugano staging was I in 16.8%, II1 in 20.8%, II2 in 10.9%, IIE in 13.9% and IV in 34.7% of cases. Twenty percent had Helicobacter pylori infection. R-CHOP-like therapy was used as first line in 96.9% of the patients. A complete response was achieved in 80% after first line therapy. At 3-years of follow-up (FU), 54% were in complete remission. The mean FU time was 73.6 months. Median overall survival and median progression free survival were not reached. We identified seven factors with negative impact in survival: age above 65 years-old (p < 0.01), ECOG 2-3 (p < 0.01), B symptoms (p = 0.001), bulky disease (p = 0.003), IPI 3-4 (p = 0.001), more than 3 treatment lines (p < 0.01), absence of response to first line treatment (p < 0.01). This study demonstrates that gastric DLBCL is a potentially curable disease with R-CHOP-like therapy, entailing long term survival and comparing well with other published series.

Identification of TNFR2 and IL-33 as therapeutic targets in localized fibrosis.

Dissecting the molecular landscape of fibrotic disease, a major unmet need, will inform the development of novel treatment strategies to target disease progression and identify desperately needed therapeutic targets. Here, we provide a detailed single-cell analysis of the immune landscape in Dupuytren's disease, a localized fibrotic condition of the hand, and identify a pathogenic signaling circuit between stromal and immune cells. We demonstrate M2 macrophages and mast cells as key cellular sources of tumor necrosis factor (TNF) that promotes myofibroblast development. TNF acts via the inducible TNFR2 receptor and stimulates interleukin-33 (IL-33) secretion by myofibroblasts. In turn, stromal cell IL-33 acts as a potent stimulus for TNF production from immune cells. Targeting this reciprocal signaling pathway represents a novel therapeutic strategy to inhibit the low-grade inflammation in fibrosis and the mechanism that drives chronicity.

Discontinuation of tyrosine kinase inhibitors in CML patients in real-world clinical practice at a single institution.

BACKGROUND: Most patients with chronic myeloid leukemia (CML) treated with tyrosine kinase inhibitors (TKIs) will relapse if treatment is withdrawn, but various trials have recently demonstrated that a significant proportion of patients who achieved a stable and deep molecular response (DMR) can stop therapy without relapsing. However, most information on treatment cessation was obtained from clinical trials with strict recruiting criteria. METHODS: We evaluated the outcome of 25 patients with CML that discontinued TKI therapy in our institute in real-world clinical practice. RESULTS: Of the 25 patients, 76% discontinued therapy in sustained deep molecular response (SDMR) and 24% were in unsustained DMR (UDMR). Discontinuation of therapy due to adverse effects was observed in 5 and 50% of the patients in the SDMR and UDMR groups, respectively. After TKI discontinuation, patients were followed for a median of 24 months. At the time of this analysis, 56% patients had a molecular relapse after a median of 4 months. SDMR and longer treatment duration were associated with lower probability of molecular relapse: 25% in SDMR patients with TKI treatment > 96 months and 85% in UDMR patients with TKI treatment ≤96 months. All relapsed patients promptly resumed TKI therapy and regained at least major molecular response (MMR). CONCLUSIONS: Our results suggest that TKI discontinuation is safe outside clinical trials and particularly effective in CML patients who are in SDMR with longer TKI treatment duration.

Fully reduced HMGB1 accelerates the regeneration of multiple tissues by transitioning stem cells to GAlert.

A major discovery of recent decades has been the existence of stem cells and their potential to repair many, if not most, tissues. With the aging population, many attempts have been made to use exogenous stem cells to promote tissue repair, so far with limited success. An alternative approach, which may be more effective and far less costly, is to promote tissue regeneration by targeting endogenous stem cells. However, ways of enhancing endogenous stem cell function remain poorly defined. Injury leads to the release of danger signals which are known to modulate the immune response, but their role in stem cell-mediated repair in vivo remains to be clarified. Here we show that high mobility group box 1 (HMGB1) is released following fracture in both humans and mice, forms a heterocomplex with CXCL12, and acts via CXCR4 to accelerate skeletal, hematopoietic, and muscle regeneration in vivo. Pretreatment with HMGB1 2 wk before injury also accelerated tissue regeneration, indicating an acquired proregenerative signature. HMGB1 led to sustained increase in cell cycling in vivo, and using Hmgb1-/- mice we identified the underlying mechanism as the transition of multiple quiescent stem cells from G0 to GAlert HMGB1 also transitions human stem and progenitor cells to GAlert Therefore, exogenous HMGB1 may benefit patients in many clinical scenarios, including trauma, chemotherapy, and elective surgery.

Bruton's tyrosine kinase regulates TLR7/8-induced TNF transcription via nuclear factor-κB recruitment.

Tumour necrosis factor (TNF) is produced by primary human macrophages in response to stimulation by exogenous pathogen-associated molecular patterns (PAMPs) and endogenous damage-associated molecular patterns (DAMPs) via Toll-like receptor (TLR) signalling. However, uncontrolled TNF production can be deleterious and hence it is tightly controlled at multiple stages. We have previously shown that Bruton's tyrosine kinase (Btk) regulates TLR4-induced TNF production via p38 MAP Kinase by stabilising TNF messenger RNA. Using both gene over-expression and siRNA-mediated knockdown we have examined the role of Btk in TLR7/8 mediated TNF production. Our data shows that Btk acts in the TLR7/8 pathway and mediates Ser-536 phosphorylation of p65 RelA and subsequent nuclear entry in primary human macrophages. These data show an important role for Btk in TLR7/8 mediated TNF production and reveal distinct differences for Btk in TLR4 versus TLR7/8 signalling.

A Genome-wide Association Study of Dupuytren Disease Reveals 17 Additional Variants Implicated in Fibrosis.

Individuals with Dupuytren disease (DD) are commonly seen by physicians and surgeons across multiple specialties. It is an increasingly common and disabling fibroproliferative disorder of the palmar fascia, which leads to flexion contractures of the digits, and is associated with other tissue-specific fibroses. DD affects between 5% and 25% of people of European descent and is the most common inherited disease of connective tissue. We undertook the largest GWAS to date in individuals with a surgically validated diagnosis of DD from the UK, with replication in British, Dutch, and German individuals. We validated association at all nine previously described signals and discovered 17 additional variants with p ≤ 5 × 10-8. As a proof of principle, we demonstrated correlation of the high-risk genotype at the statistically most strongly associated variant with decreased secretion of the soluble WNT-antagonist SFRP4, in surgical specimen-derived DD myofibroblasts. These results highlight important pathways involved in the pathogenesis of fibrosis, including WNT signaling, extracellular matrix modulation, and inflammation. In addition, many associated loci contain genes that were hitherto unrecognized as playing a role in fibrosis, opening up new avenues of research that may lead to novel treatments for DD and fibrosis more generally. DD represents an ideal human model disease for fibrosis research.

Southwestern Oncology Group pretreatment risk criteria as predictive or prognostic factors in acute myeloid leukemia.

Acute myeloid leukemia (AML) is a clonal hematological malignant condition and the implications of pretreatment risk criteria as predictive or prognostic factors are constantly under evaluation. With this study, the authors' intent was to characterize AML patients and to evaluate the clinical outcome associated with Southwestern Oncology Group (SWOG) coding pretreatment risk criteria/cytogenetic score. Between 2002 and 2010, 225 patients were diagnosed with AML at the Portuguese Institute of Oncology (Porto, Portugal). From this patient group, 128 patients aged <65 years were selected. The patients were treated using a combination of cytarabine and anthracycline, with the addition of cyclosporine when bone marrow dysplasia was observed. A median survival of 24 months was observed in this group. The patients were divided in subgroups according to the SWOG pretreatment risk criteria. We observed a statistically significant association of non-favorable SWOG coding with female gender [P=0.025; risk ratio (RR)=3.632, 95% confidence interval (CI): 1.113-11.852], indication for allogeneic bone marrow transplantation (P=0.023, RR=1.317, 95% CI: 1.184-1.465), complete response achievement (P=0.013, RR=1.385, 95% CI: 11.232-1.556) and relapse (P=0.048, RR=3.181, 95% CI: 10.966-10.478). Furthermore, SWOG pretreatment risk criteria also significantly affected global overall survival (OS; P=0.003) and OS at 5 years (P=0.001). A multivariate Cox regression analysis supported response to induction therapy (3-year OS: P=0.011, RR=0.385, 95% CI: 10.184-0.806; 5-year OS: P=0.012, RR=0.388, 95% CI: 10.597-1.994), consolidation (3-year OS: P=0.005, RR=0.328, 95% CI: 0.150-0.720; 5-year OS: P=0.002, RR=0.308, 95% CI: 0.144-0.657) and the diagnosis of therapy-related aml (3-year OS: P=0.016, RR=2.756, 95% CI: 0.486-1.281; 5-year OS: P=0.031, RR=2.369, 95% CI: 1.081-5.189) as prognostic factors, but this was not confirmed for SWOG pretreatment risk criteria. Therefore, we concluded that the reproducibility of the application of the SWOG pretreatment risk criteria may not be available as a prognostic factor in every acute leukemia population. However, its application as a predictive factor of response has been confirmed in our population.

Effect of non-coherent infrared light (LED, λ945 ± 20 nm) on bone repair in diabetic rats-morphometric and spectral analyses.

Phototherapy using coherent light (lasers) and non-coherent light (light-emitting diodes (LEDs)) has been investigated for the purpose of biomodulation in biological tissues. Several effects can be expected, including pain moderation, biostimulation of cellular tropism, anti-inflammatory effects, regular circulatory stimulation, and tissue repair. The aim of this study was to evaluate the effect of LED (λ945 ± 20 nm, 48 mW) therapy on the regeneration process in femoral lesions of rats (Wistar). Seven irradiation sessions were held, with a 48-h interval between sessions. The animals were euthanised 14, 21, and 28 days after surgery. Bone samples were analysed by histomorphometry, micro X-ray fluorescence spectroscopy, scanning electron microscopy, and optical densitometry. The results demonstrated the effective positive influence of low-intensity LED therapy using the near-infrared region on the tissue repair process in diabetic animals, especially in the early stages of repair (14 and 21 days after surgery). It can be concluded that LED therapy positively influences bone formation in the early stages of the bone repair process in non-diabetic and diabetic animals, without causing changes in the optical density and volume of tissue in the final stages. No influence of LED therapy was observed on the percentage of calcium, percentage of phosphorus, Ca/P ratio, or optical mineral density in non-diabetic animals. However, increased mineral concentration was evident in the diabetic animals treated with the LED during the repair process.

Effect of therapy-related acute myeloid leukemia on the outcome of patients with acute myeloid leukemia.

Therapy-related acute myeloid leukemia (t-AML) is a rare and almost always fatal late side effect of antineoplastic treatment involving chemotherapy, radiotherapy or the two combined. The present retrospective study intended to characterize t-AML patients that were diagnosed and treated in a single referral to an oncological institution in North Portugal. Over the past 10 years, 231 cases of AML were diagnosed and treated at the Portuguese Institute of Oncology of Porto, of which 38 t-AML cases were identified. Data regarding the patient demographics, primary diagnosis and treatment, age at onset of therapy-related myeloid neoplasm, latency time of the neoplasm, cytogenetic characteristics, AML therapy and outcome were collected from medical records. A previous diagnosis with solid tumors was present in 28 patients, and 10 patients possessed a history of hematological conditions, all a lymphoproliferative disorder. Breast cancer was the most frequent solid tumor identified (39.5% of all solid tumors diagnosed). The mean latency time was 3 years. In the present study, t-AML patients were older (P<0.001) and more frequently carried cytogenetic abnormalities (P=0.009) compared with de novo AML patients. The overall survival time was observed to be significantly poorer among individuals with t-AML (P<0.001). However, in younger patients (age, <50 years) there was no difference between the overall survival time of patients with t-AML and those with de novo AML (P=0.983). Additionally, patients with promyelocytic leukemia possess a good prognosis, even when AML occurs as a secondary event (P=0.98). To the best of our knowledge, the present study is the first to evaluate t-AML in Portugal and the results are consistent with the data published previously in other populations. The present study concludes that although t-AML demonstrates a poor prognosis, this is not observed among younger patients or promyelocytic leukemia patients.

Effects of Bleaching Agents Combined with Regular and Whitening Toothpastes on Surface Roughness and Mineral Content of Enamel.

OBJECTIVE: The purpose of this study was to evaluate surface roughness and changes in the composition of enamel submitted to different bleaching protocols and toothbrushing with regular and whitening toothpastes. BACKGROUND DATA: Bleaching treatment could promote morphological and chemical changes in enamel surface. METHODS: Enamel blocks were randomized into nine groups (n=10) according to the bleaching treatment (no bleaching, control group; 6% hydrogen peroxide, HP; or 10% carbamide peroxide, CP) and toothpaste used (placebo, PL; regular, R; or whitening dentifrice, W). Bleaching was performed according to manufacturers' instructions and all groups were submitted to 30,000 cycles of simulated toothbrushing with toothpaste (PL, R, or W). Mineral content evaluation and enamel roughness were evaluated initially (T1), after bleaching (T2), and after toothbrushing (T3), using an energy-dispersive micro X-ray fluorescence spectrometer and profilometry, respectively. Data were statistically analyzed with two way ANOVA, Tukey, and Dunnett tests (5%). RESULTS: Enamel surface roughness was influenced by bleaching and toothbrushing. Surface roughness increased for the groups that brushed with the placebo dentifrice (CP+PL, HP+PL, C+PL) and for the control group that brushed with whitening dentifrice (C+W). Enamel Ca/P ratio decreased after bleaching, but toothbrushing, regardless of the dentifrice used, did not reduce the enamel mineral content. CONCLUSIONS: The bleaching treatment resulted in a decrease of enamel mineral content, but the studied dentifrices did not contribute to surface mineral loss.

Low-dose TNF augments fracture healing in normal and osteoporotic bone by up-regulating the innate immune response.

The mechanism by which trauma initiates healing remains unclear. Precise understanding of these events may define interventions for accelerating healing that could be translated to the clinical arena. We previously reported that addition of low-dose recombinant human TNF (rhTNF) at the fracture site augmented fracture repair in a murine tibial fracture model. Here, we show that local rhTNF treatment is only effective when administered within 24 h of injury, when neutrophils are the major inflammatory cell infiltrate. Systemic administration of anti-TNF impaired fracture healing. Addition of rhTNF enhanced neutrophil recruitment and promoted recruitment of monocytes through CCL2 production. Conversely, depletion of neutrophils or inhibition of the chemokine receptor CCR2 resulted in significantly impaired fracture healing. Fragility, or osteoporotic, fractures represent a major medical problem as they are associated with permanent disability and premature death. Using a murine model of fragility fractures, we found that local rhTNF treatment improved fracture healing during the early phase of repair. If translated clinically, this promotion of fracture healing would reduce the morbidity and mortality associated with delayed patient mobilization.

The Cdc42 guanine nucleotide exchange factor FGD6 coordinates cell polarity and endosomal membrane recycling in osteoclasts.

The initial step of bone digestion is the adhesion of osteoclasts onto bone surfaces and the assembly of podosomal belts that segregate the bone-facing ruffled membrane from other membrane domains. During bone digestion, membrane components of the ruffled border also need to be recycled after macropinocytosis of digested bone materials. How osteoclast polarity and membrane recycling are coordinated remains unknown. Here, we show that the Cdc42-guanine nucleotide exchange factor FGD6 coordinates these events through its Src-dependent interaction with different actin-based protein networks. At the plasma membrane, FGD6 couples cell adhesion and actin dynamics by regulating podosome formation through the assembly of complexes comprising the Cdc42-interactor IQGAP1, the Rho GTPase-activating protein ARHGAP10, and the integrin interactors Talin-1/2 or Filamin A. On endosomes and transcytotic vesicles, FGD6 regulates retromer-dependent membrane recycling through its interaction with the actin nucleation-promoting factor WASH. These results provide a mechanism by which a single Cdc42-exchange factor controlling different actin-based processes coordinates cell adhesion, cell polarity, and membrane recycling during bone degradation.

Pharmacogenetic considerations for non-Hodgkin's lymphoma therapy.

INTRODUCTION: Chemotherapy is the current standard treatment for hematological malignancies for both curative and palliative purposes. Unfortunately, in the current treatment scenario chemotherapy resistance is an issue that is know to lead to a relapse in cancer. The multidrug resistance 1 (MDR1) gene is often involved in drug resistance and, so far, the best studied mechanism of resistance relates to the level of P-glycoprotein (P-gp) expression on cancer cells; however, correlation with single nucleotide polymorphism (SNP) in the MDR1 gene has also been observed via a number of different mechanisms that interfere with function and expression of P-gp. AREAS COVERED: This article describes the influence of P-gp expression and SNP on the MDR1 gene in non-Hodgkin's lymphoma (NHL) and their effect on both its risk and outcome. The authors also provide a brief summary of the more important therapeutic options, which aim to overcome this drug resistance mechanism, and discuss their known mechanisms of action. EXPERT OPINION: There is evidence pertaining to an association between the outcome of NHL and P-gp expression. However, the authors emphasize the need for more studies to reinforce this evidence. Furthermore, there is a definite need for the therapeutic targets, which provide tumor cellular lines of interest, to be tested in humans, in order to better evaluate their toxicity and overall effect on the outcome. The ultimate aim of this research is to develop specifically designed therapies that are tailored to the intrinsic characteristics of specific patients.

Fibers from fruit by-products enhance probiotic viability and fatty acid profile and increase CLA content in yoghurts.

This study evaluated the effect of the supplementation of total dietary fiber from apple, banana or passion fruit processing by-products on the post-acidification, total titratable acidity, bacteria counts and fatty acid profiles in skim milk yoghurts co-fermented by four different probiotics strains: Lactobacillus acidophilus L10 and Bifidobacterium animalis subsp. lactis BL04, HN019 and B94. Apple and banana fibers increased the probiotic viability during shelf-life. All the fibers were able to increase the short chain and polyunsaturated fatty acid contents of yoghurts compared to their respective controls. A synergistic effect between the type of fiber and the probiotic strain on the conjugated linoleic acid content was observed, and the amount of α-linolenic acid was increased by banana fiber. The results of this study demonstrate, for the first time, that fruit fibers can improve the fatty acid profile of probiotic yoghurts and point out the suitability of using fibers from fruit processing the by-products to develop new high value-added fermented dairy products.

Erosion effects on chemical composition and morphology of dental materials and root dentin.

PURPOSE: This work aims to study the erosion on restorative materials and on surrounding dentin. Fifty root dentin samples were obtained from bovine incisors. METHODS: Twenty samples were not restored and thirty received cavity preparations. Samples were assigned to five groups: G1, G2: sound dentin (D); G3: composite resin (CR); G4: resin-modified glass-ionomer cement (RMGIC); G5: glass-ionomer cement (GIC). The samples of groups 2-5 were submitted to six cycles (demineralization-remineralization). Samples were analyzed by micro energy-dispersive X-ray fluorescence spectrometry (µ-EDXRF) and by scanning electron microscopy (SEM). RESULTS: Mineral loss was greater in G2 samples than in RMGI > CR > GIC > D (control). SEM images showed pronounced dentin demineralization in groups 2 and 4. The acid erosion has a significant effect on mineral loss (Ca and P) of root dentin without restoration. CONCLUSIONS: Composite resin had the best chemical resistance to erosion among all the materials. Fluoride contained in GIC seemed to cause some protection, however, with material degradation. Chemical interaction of tooth-colored dental materials with root dentin could be assessed by µ-EDXRF.

Energy dispersive X-ray spectrometry study of the protective effects of fluoride varnish and gel on enamel erosion.

BACKGROUND: Dental erosion is a risk factor for dental health, introduced by today's lifestyle. Topical fluoride applications in the form of varnishes and gel may lead to deposition of fluoride on enamel. PURPOSE: This in vitro study aimed to evaluate the effect of two fluoride varnishes and one fluoride gel on the dissolution of bovine enamel by acids. METHODS: Enamel samples (72) were divided (n = 8): artificial saliva (control-G1), Pepsi Twist® (G2), orange juice (G3), Duraphat® + Pepsi Twist® (G4), Duraphat® + orange juice (G5), Duofluorid® + Pepsi Twist® (G6), Duofluorid® + orange juice (G7), fluoride gel + Pepsi Twist® (G8), and fluoride gel + orange juice (G9). Fluoride gel was applied for 4 min and the varnishes were applied and removed after 6 h. The samples were submitted to six cycles (demineralization: Pepsi Twist® or orange juice, 10 min; remineralization: saliva, 1 h). Samples were analyzed by energy-dispersive X-ray fluorescence (144 line-scanning). RESULTS: The amount of Ca and P decreased significantly in the samples of G2 and G3, and the Ca/P ratio decreased in G3. Mineral gain (Ca) was greater in G9 samples than in G4 > G3 > G5 > G1, and (P) greater in G7 samples than in G9 > G4-6 > G2-3. CONCLUSIONS: The protective effect of Duofluorid® was significantly lower than fluoride gel against orange juice. The fluoride varnishes can interfere positively with the dissolution of dental enamel in the presence of acidic beverages. Fluoride gel showed the best protection level to extrinsic erosion with low costs.

FT-Raman and energy dispersive X-ray fluorescence spectrometric analyses of enamel submitted to 38% hydrogen peroxide bleaching, an acidic beverage, and simulated brushing.

OBJECTIVE: This study aimed to investigate the effects on enamel surface treated with hydrogen peroxide bleaching and acidic soft drink immersion and/or brushing with whitening dentifrices. MATERIALS AND METHODS: Fifty-six standardized enamel slabs obtained from labial surfaces of bovine incisors were used. Enamel slabs were ground flat, polished, and randomly assigned to one of seven treatment groups: (1) control, in which no treatment was performed, (2) soft drink immersion, (3) 38% hydrogen peroxide bleaching, (4) simulated toothbrushing with whitening dentifrice, (5) soft drink immersion and bleaching, (6) soft drink immersion, bleaching, and toothbrushing, and (7) bleaching and toothbrushing. The mineral concentration of enamel surfaces was determined before and after treatments by means of Fourier transform (FT)-Raman spectroscopy and energy dispersive x-ray fluorescence spectrometry (EDXRF). Data were statistically analyzed by ANOVA and Tukey test (p < 0.05). RESULTS: Raman spectroscopy results indicated that enamel mineral content decreased after all treatments except in group 1, whereas EDXRF results exhibited mineral decrease in groups 3, 4, 5, and 7. CONCLUSION: Bleaching alone or in combination with soft drink immersion and brushing decreases enamel mineral content.