Detection of Aeromonas salmonicida subsp. salmonicida infection in zebrafish by labelling bacteria with GFP and a fluorescent probe based on the siderophore amonabactin.

Zebrafish (Danio rerio) is an excellent model to study bacterial infections in fish and their treatment. We used zebrafish as a model of infection for Aeromonas salmonicida subsp. salmonicida (hereinafter A. salmonicida), the causative agent of fish furunculosis. The infection process of A. salmonicida was studied by immersion of zebrafish larvae in 2 different doses of the bacteria and the fish mortality was monitored for three days. The bacterium caused a high mortality (65 %) in zebrafish larvae only when they were exposed to a high bacterial concentration (107 bacterial cells/mL). To evaluate the use of fluorescence microscopy to follow A. salmonicida infection in vivo, two different fluorescent strains generated by labeling an A. salmonicida strain with either, the green fluorescent protein (GFP), or with a previously reported siderophore amonabactin-sulforhodamine B conjugate (AMB-SRB), were used. The distribution of both labeled bacterial strains in the larvae tissues was evaluated by conventional and confocal fluorescence microscopy. The fluorescent signal showed a greater intensity with the GFP-labeled bacteria, so it could be observed using conventional fluorescence microscopy. Since the AMB-SRB labeled bacteria showed a weaker signal, the larvae were imaged using a laser scanning confocal microscope after 48 h of exposure to the bacteria. Both fluorescent signals were mainly observed in the larvae digestive tract, suggesting that this is the main colonization route of zebrafish for waterborne A. salmonicida. This is the first report of the use of a siderophore-fluorophore conjugate to study a bacterial infection in fish. The use of a siderophore-fluorophore conjugate has the advantage that it is a specific marker and that does not require genetic manipulation of the bacteria.

Concurrent progressive multifocal leukoencephalopathy and central nervous system infiltration by multiple myeloma: A case report.

Progressive multifocal leukoencephalopathy rarely occurs in patients with multiple myeloma. Intracranial central nervous system invasion is also an uncommon event in multiple myeloma, occurring in less than 1% of cases. We describe herein an exceptional case of coexisting progressive multifocal leukoencephalopathy and intraparenchymal central nervous system myeloma infiltration. A 73-year-old woman with relapsed multiple myeloma was treated with 15 cycles of lenalidomide and dexamethasone, but therapy had to be stopped because of a hip fracture after a fall. During hospitalization, the patient developed progressive multifocal leukoencephalopathy caused by John Cunningham virus, and a prominent intra-parenchymal CD138-positive infiltrate was detected. VDJ rearrangements of the immunoglobulin heavy chain gene and the mutational profile of plasma cells in bone marrow at the time of diagnosis and in brain biopsy after progression were analyzed by next generation sequencing, showing genetic differences between medullary and extramedullary myeloma cells. The role of long-term treatment with lenalidomide and dexamethasone in the development progressive multifocal leukoencephalopathy or intraparenchymal central nervous system myeloma infiltration remains unknown. However, our results suggest that both events may have arisen as a consequence of treatment-related immunosuppression. Thus, an appropriate clinical approach compatible with the simultaneous treatment of progressive multifocal leukoencephalopathy and multiple myeloma should be developed.

Detection of Epstein-Barr virus DNAemia after lung transplantation and its potential relationship with the development of post-transplant complications.

BACKGROUND: Recent studies suggest that Epstein-Barr virus DNAemia (EBVd) may act as a surrogate marker of post-transplant immunosuppression. This hypothesis has not been tested so far in lung transplant (LT) recipients. METHODS: We included 63 patients undergoing lung transplantation at our center between October 2008 and May 2013. Whole blood EBVd was systematically assessed by real-time polymerase chain reaction assay on a quarterly basis. The occurrence of late complications (overall and opportunistic infection [OI] and chronic lung allograft dysfunction [CLAD]) was analyzed according to the detection of EBVd within the first 6 months post transplantation. RESULTS: Any EBVd was detected in 30 (47.6%) patients. Peak EBVd was higher in patients with late overall infection (2.23 vs. 1.73 log10 copies/mL; P = 0.026) and late OI (2.39 vs. 1.74 log10 copies/mL; P = 0.004). The areas under receiver operating characteristic curves for predicting both events were 0.806 and 0.871 respectively. The presence of an EBVd ≥2 log10 copies/mL during the first 6 months post transplantation was associated with a higher risk of late OI (adjusted hazard ratio [aHR] 7.92; 95% confidence interval [CI] 2.10-29.85; P = 0.002). Patients with detectable EBVd during the first 6 months also had lower CLAD-free survival (P = 0.035), although this association did not remain statistically significant in the multivariate analysis (aHR 1.26; 95% CI 0.87-5.29; P = 0.099). CONCLUSIONS: Although preliminary in nature, our results suggest that the detection of EBVd within the first 6 months after transplantation is associated with the subsequent occurrence of late OI in LT recipients.

Could polymerase chain reaction tests on conjunctival swabs be useful to diagnose herpetic keratitis?

INTRODUCTION: Diagnosis of HSV-1 keratitis (HK) is frequently based on clinical findings. Invasive specimens (corneal scrapings, biopsies) are required for microbiological diagnosis. METHODS: Corneal scrapings and conjunctival swabs were collected on patients with/without clinical suspicion of HK from 2007 to 2012. RESULTS: The sensitivity, specificity, positive and negative predictive values for conjunctival swabs by PCR was 77.8, 92.1, 84.4 and 88.3, respectively. DISCUSSION: Conjunctival swabs by PCR may help in the diagnosis of HK, despite the limited sensitivity.

First confirmation of importation and transmission in Spain of the newly identified SARS-CoV-2 B.1.1.7 variant.

INTRODUCTION: A newly identified SARS-CoV-2 variant, VOC202012/01 originating lineage B.1.1.7, recently emerged in the United Kingdom. The rapid spread in the UK of this new variant has caused other countries to be vigilant. MATERIAL AND METHODS: We based our initial screening of B.1.1.7 on the dropout of the S gene signal in the TaqPath assay, caused by the 69/70 deletion. Subsequently, we confirmed the B.1.1.7 candidates by whole genome sequencing. RESULTS: We describe the first three imported cases of this variant from London to Madrid, subsequent post-arrival household transmission to three relatives, and the two first cases without epidemiological links to UK. One case required hospitalization. In all cases, drop-out of gene S was correctly associated to the B.1.1.7 variant, as all the corresponding sequences carried the 17 lineage-marker mutations. CONCLUSION: The first identifications of the SARS-CoV-2 B.1.1.7 variant in Spain indicate the role of independent introductions from the UK coexisting with post-arrival transmission in the community, since the early steps of this new variant in our country.

Role of molecular diagnostics in the clinical management of SARS-CoV-2 infection: advantages and drawbacks.

The diagnosis of SARS-CoV-2 is based on the use of nucleic acid amplification tests (NAAT), especially rRT-PCR. The latter also allows us to quickly identify variants of concern. However, its use in follow-up of patients and the correlation between Ct value and the viability of the virus is controversial.

First confirmation of importation and transmission in Spain of the newly identified SARS-CoV-2 B.1.1.7 variant.

INTRODUCTION: A newly identified SARS-CoV-2 variant, VOC202012/01 originating lineage B.1.1.7, recently emerged in the United Kingdom. The rapid spread in the UK of this new variant has caused other countries to be vigilant. MATERIAL AND METHODS: We based our initial screening of B.1.1.7 on the dropout of the S gene signal in the TaqPath assay, caused by the 69/70 deletion. Subsequently, we confirmed the B.1.1.7 candidates by whole genome sequencing. RESULTS: We describe the first three imported cases of this variant from London to Madrid, subsequent post-arrival household transmission to three relatives, and the two first cases without epidemiological links to UK. One case required hospitalization. In all cases, drop-out of gene S was correctly associated to the B.1.1.7 variant, as all the corresponding sequences carried the 17 lineage-marker mutations. CONCLUSION: The first identifications of the SARS-CoV-2 B.1.1.7 variant in Spain indicate the role of independent introductions from the UK coexisting with post-arrival transmission in the community, since the early steps of this new variant in our country.

Expression of heterochromatin protein 1 in the primary tumor of breast cancer patients in the presence or absence of occult metastatic cells in the bone marrow.

Gene silencing may occur in breast cancer samples from patients presenting with occult metastatic cells in the bone marrow and one mechanism regulating gene suppression is heterochromatin formation. We have studied whether members of the heterochromatin protein 1 family (HP1Hs alpha, HP1Hs beta and HP1Hs gamma), which take part in chromatin packaging and gene expression regulation, were differentially expressed in tumors from patients with and without occult metastatic cells in their bone marrow. Tumor samples and bone marrow aspirates were obtained from 37 breast cancer patients. Median age was 63 years and 68% of the patients presented with clinical stage I/II disease. Presence of occult metastatic cells in bone marrow was detected through keratin-19 expression by nested RT-PCR in samples from 20 patients (54.1%). The presence of occult metastatic cells in bone marrow was not associated with node involvement, histological grade, estrogen receptor and ERBB2 immunoexpression. Relative gene expression of HP1Hs alpha, HP1Hs beta and HP1Hs gamma was determined by realtime RT-PCR and did not vary according to the presence of occult metastatic cells in bone marrow. In addition, the combined expression of these three transcripts could not be used to classify samples according to the presence of bone marrow micrometastasis. Our work indicates that regulation of heterochromatin formation through HP1 family members may not be the sole mechanism implicated in the metastatic process to the bone marrow.

Orthotopic tumorgrafts in nude mice as a model to evaluate calcitriol effects in breast cancer.

OBJECTIVES: Calcitriol antiproliferative effects were observed in xenografts of breast cancer cell lines, however they were not yet investigated in tumorgrafts, consisting of freshly collected breast cancer samples xenografted into animals. To establish a tumorgraft model, from freshly collected breast cancer samples, which were directly implanted in nude mice, to study calcitriol effects. METHODS: Breast cancer samples collected from 12 patients were orthotopically implanted into nude mice. Animals were treated with weekly intratumoral injections of calcitriol 3 µg/Kg, which was previously shown to induce peak serum calcitriol levels in the predicted therapeutic range. RESULTS: Success engraftment rate was 25%. Tumorgrafts were established from aggressive (HER2 positive or histological grade 3) highly proliferative samples and original tumor characteristics were preserved. Calcitriol highly induced its target gene, CYP24A1, indicating that the genomic vitamin D pathway is active in tumorgrafts. However, no differences in the expression of proliferation and apoptosis markers (BrdU incorporation, Ki67, CDKN1A, CDKN1B, BCL2 expression) were observed in these highly proliferative tumor samples. CONCLUSIONS: Tumorgrafts seem a promising model to explore other calcitriol doses and regimens, considering the heterogeneity of the disease and microenvironment interactions.

Gene expression profiling of clinical stages II and III breast cancer.

Clinical stage (CS) is an established indicator of breast cancer outcome. In the present study, a cDNA microarray platform containing 692 genes was used to identify molecular differences between CSII and CSIII disease. Tumor samples were collected from patients with CSII or CSIII breast cancer, and normal breast tissue was collected from women without invasive cancer. Seventy-eight genes were deregulated in CSIII tumors and 22 in CSII tumors when compared to normal tissue, and 20 of them were differentially expressed in both CSII and CSIII tumors. In addition, 58 genes were specifically altered in CSIII and expression of 6 of them was tested by real time RT-PCR in another cohort of patients with CSII or CSIII breast cancer and in women without cancer. Among these genes, MAX, KRT15 and S100A14, but not APOBEC3G or KRT19, were differentially expressed on both CSIII and CSII tumors as compared to normal tissue. Increased HMOX1 levels were detected only in CSIII tumors and may represent a molecular marker of this stage. A clear difference in gene expression pattern occurs at the normal-to-cancer transition; however, most of the differentially expressed genes are deregulated in tumors of both CS (II and III) compared to normal breast tissue.

Experimental study of the connections of the preglomerular nuclei and corpus mamillare in the rainbow trout, Oncorhynchusmykiss.

The preglomerular complex of trout consists of the anterior (aPGN) and medial (mPGN) preglomerular nuclei and the corpus mamillare (CM). In order to improve knowledge on this complex, we applied a lipophilic neuronal tracer (DiI) to the three nuclei. These nuclei received afferents from the medial part of the dorsal telencephalic area (Dm), the ventral part of the ventral telencephalic area (Vv), the preoptic nucleus, the periventricular layer of the rostral optic tectum and the central posterior thalamic nucleus. The aPGN also received numerous toral projections and, sent efferents to the anterior tuberal nucleus. In addition, both the aPGN and the mPGN nuclei gave rise to efferents to the dorsal region of the dorsal telencephalic area (Dd), whereas the medial preglomerular nucleus and the CM sent fibers to the torus lateralis and the diffuse nucleus, as confirmed by reciprocal labeling. A small mPGN/CM subgroup projected to the optic tectum. These results suggest close functional inter-relationship between the trout preglomerular complex and two telencephalic regions (Dm and Vv). In addition, all nuclei of the complex receive preoptic, tectal and dorsal thalamic afferents, whereas the aPGN and mPGN are related with acoustic-lateral ascending pathways, and the mPGN and CM with the central region of the dorsal telencephalic area and visceral/gustatory pathways.

Everolimus-based immunosuppression therapy for BK virus nephropathy.

BACKGROUND: Mammalian target of rapamycin inhibitors (mTOR-i) have been proposed as possible immunosuppressants of choice in BK virus nephropathy (BKN) because of their antiviral capacity. On this basis, in 2007, our Service proposed a conversion to everolimus (EVE)-based therapy from calcineurin inhibitors with an anti-calcineurin-free therapy protocol in those patients diagnosed of BKN. METHODS: A prospective, single-center case series study was performed. Fifteen cases of BKN were diagnosed from 2007 to the end of 2010. According to our protocol, immunosuppressant treatment was modified in 9 of these patients with suspension of mycophenolate and conversion from tacrolimus to EVE. RESULTS: The renal function achieved by our patients after the transplantation was excellent. Mean serum creatinine (sCr) achieved was 1.16 ± 0.2 mg/dL. Evolution of the renal function after BKN diagnosis and conversion to mTOR-i was positive in all the patients. sCr on diagnosis was 1.85 ± 0.22 mg/dL, sCr at the point in time of conversion to EVE was 2 ± 0.21 mg/dL, and final sCr of the follow-up was 1.6 ± 0.39 mg/dL (P = .05). BK viremia became negative in 5 of our patients and decreased more than 95% in the remaining 4. None of the patients had an acute rejection episode after the change of immunosuppressant. CONCLUSIONS: Conversion to mTOR-i-based therapy could provide an added benefit in BKN and could be an effective strategy for the decrease of the viremia and increase of graft survival in selected patients.

Influence of cytomegalovirus infection in the development of cardiac allograft vasculopathy after heart transplantation.

BACKGROUND: Cardiac allograft vasculopathy (CAV) is a major cause of long-term morbidity and mortality after heart transplantation (HTx), whose relationship with CMV infection is uncertain. This study evaluated the influence of CMV infection in the development of CAV. METHODS: We enrolled 166 consecutive HTx recipients who underwent their first transplant from January 1995 to July 2002. All patients received 14 days of intravenous ganciclovir and were prospectively monitored for CMV infection during the first year after HTx. CAV was diagnosed by coronary angiography performed at 1, 5, and 10 years after HTx, following the new criteria of the International Society for Heart and Lung Transplantation. We collected all variables potentially related with the development of CAV. Risk factors were studied using a complementary log-log model. RESULTS: After a median follow-up of 11 years (range, 1-17 years), 72 patients (43%) developed CAV (63.8% CAV(1), 15.2% CAV(2), 20.8% CAV(3)). Symptoms secondary to CAV were present in 32% of these patients, and 8% died because of it. In the regression multivariate analysis, independent variables associated with the development of CAV were donor age (hazard ratio [HR], 1.028; 95% confidence interval [CI], 1.002-1.053; p < 0.028), presence of cellular acute rejection ≥ 2R (HR, 1.764; 95% CI, 1.011-3.078; p < 0.0414), CMV infection (HR, 2.334; 95% CI, 1.043-5.225; p < 0.0354), and not having been treated with a calcium channel blocker (HR, 0.472; 95% CI, 0.275-0.811; p < 0.0055). CONCLUSIONS: Standardized angiographic criteria show CMV infection is associated with the development of CAV.

Expression of vitamin D receptor (VDR) in HL-60 cells is differentially regulated during the process of differentiation induced by phorbol ester, retinoic acid or interferon-gamma.

The effects of three inducers of differentiation, phorbol myristate acetate (PMA), retinoic acid (RA) and interferon-gamma (IFN-gamma), on the temporal regulation of vitamin D receptor (VDR) expression in HL-60 cells were analyzed by Northern blotting and immunofluorescence assays. VDR, at the protein level, expressed by 81% of uninduced cells, was reduced to 57% after 48 h of PMA or 96 h of RA treatment, preceded by growth inhibition and cell differentiation, evaluated by CD11b expression. Sorted CD11b positive cells in G0/G1 phase exhibited 53% the VDR content of CD11b negative cells (distributed throughout the cell cycle). PMA also induced an increase in PKC beta and PKC alpha mRNA and protein. Simultaneous exposure to PMA and sphingosine blocked stimulation of CD11b and PKC expression without affecting growth arrest and VDR down regulation. Similar effects were observed during sphingosine treatment. In IFN-gamma differentiated cells, the proportion of cells in G0/G1 phase was unchanged and VDR protein was unaltered as compared to uninduced cells. Control cells in G0/G1 expressed less VDR than cells in S and G2/M phases (74% and 59% respectively). All results suggest that in HL-60 cells, reduction of VDR expression is related to growth inhibition rather than to the differentiation process.

Influence of bacterial strains on bone infection.

Experiments were performed on 120 rabbits to compare the probability of infection after bone surgery without an implant, with polymethylmethacrylate, and with autografts. Staphylococcus aureus phage type 94/96, isolated from a human osteomyelitis, was instilled into the intramedullar cavity after reaming of the femoral canal and before insertion of the implant. The different 50% infective doses were determined for each of the groups for comparative purposes. The bacterial concentrations required to produce infection in femora without an implant were two times less than those necessary in femora implanted with polymethylmethacrylate. The bone graft required bacterial concentrations nine times less than those necessary to infect femora containing polymethylmethacrylate and four times less than those required to infect femora without an implant. The results presented here confirm that the susceptibility to infection in orthopaedic surgery is not only material dependent but also bacteria dependent.

Influence of metal implants on infection. An experimental study in rabbits.

We implanted cylinders of cobalt-chrome or titanium, with smooth or porous surfaces, into rabbit bones which had been inoculated with suspensions of Staphylococcus aureus in various doses. The bacterial concentration required to produce infection of porous-coated titanium implants was 2.5 times smaller than that necessary to infect implants with polished surfaces. Porous-coated cobalt-chromium implants required bacterial concentrations that were 40 times smaller than those needed to infect implants with polished surfaces, and 15 times smaller than those required to infect porous-coated titanium implants. The other advantages and disadvantages of the various implants, such as improved osseointegration, larger ion-release surfaces, surface wear and relative stiffness, must be weighed against the higher infection rates in the porous-coated implants, and particularly in the cobalt-chromium implants.

Normal HC11 and ras-transformed mouse mammary cells are resistant to the antiproliferative effects of retinoic acid.

The objective of the present study was to determine the effects of retinoic acid on the growth of the mouse mammary cells HC11 and HC11ras, which are a model for in vitro breast cancer progression. The expression of the two classes (RARs and RXRs) of retinoic acid receptor mRNAs was determined by Northern blot analysis. Receptor functional integrity was determined by testing whether RAR mRNA could be induced by retinoic acid. The effects of a 72-h exposure to 50 M 13-cis retinoic acid on HC11 and HC11ras cell proliferation and HC11 cell differentiation were investigated by flow cytometric cell cycle analysis, and by determination of -casein mRNA expression, respectively. The possibility that retinoic acid would induce the expression of the vitamin D receptor and synergize with vitamin D, a known inhibitor of HC11 cell growth, was also investigated. HC11 cells expressed higher mRNA levels of both RAR a and RAR g when compared to HC11ras cells. In contrast, RAR , as well as RXR a, and g expression was low in both HC11 and HC11ras cells. In addition, RAR mRNA was induced by retinoic acid treatment in both cells. In spite of these observations, no effects were seen on cell proliferation or differentiation upon exposure to retinoic acid. Neither vitamin D receptor induction nor synergy with vitamin D on growth inhibition was observed. We conclude that the RAR expression profile could be related to the transformed state in HC11ras cells and that the retinoic acid resistance observed merits further investigation.

Antiproliferative effects of 1,25-dihydroxyvitamin D3 on breast cells: a mini review.

The hormone 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3), the active form of vitamin D3, is an important regulator of calcium homeostasis, exerts antiproliferative effects on various cell systems and can induce differentiation in some kinds of hematopoietic cells. These effects are triggered by its receptor, vitamin D receptor (VDR), a phosphoprotein member of the nuclear receptor superfamily, which functions as a transcriptional factor. VDR binds as a heterodimer with retinoid X receptor (R X R) to hexameric repeats, characterized as vitamin D-responsive elements present in the regulatory region of target genes such as osteocalcin, osteopontin, calbindin-D28K, calbindin-D9K, p21WAF1/CIP1, TGF-beta2 and vitamin D 24-hydroxylase. Many factors such as glucocorticoids, estrogens, retinoids, proliferation rate and cell transformation can modulate VDR levels. VDR is expressed in mammary tissue and breast cancer cells, which are potential targets to hormone action. Besides having antiproliferative properties, vitamin D might also reduce the invasiveness of cancer cells and act as an anti-angiogenesis agent. All of these antitumoral features suggest that the properties of vitamin D could be explored for chemopreventive and therapeutic purposes in cancer. However, hypercalcemia is an undesirable side effect associated with pharmacological doses of 1,25-(OH)2D3. Some promising 1,25-(OH)2D3 analogs have been developed, which are less hypercalcemic in spite of being potent antiproliferative agents. They represent a new field of investigation.

Differential regulation of vitamin D receptor expression in distinct leukemic cell lines upon phorbol ester-induced growth arrest.

A close correlation between vitamin D receptor (VDR) abundance and cell proliferation rate has been shown in NIH-3T3 fibroblasts, MCF-7 breast cancer and in HL-60 myeloblastic cells. We have now determined if this association occurs in other leukemic cell lines, U937 and K562, and if VDR content is related to c-myc expression, which is also linked to cell growth state. Upon phorbol myristate acetate (PMA) treatment, cells from the three lineages (HL-60, U937 and K562) differentiated and expressed specific surface antigens. All cell lines analyzed were growth inhibited by PMA and the doubling time was increased, mainly due to an increased fraction of cells in the G0/G1 phase, as determined by flow cytometry measurements of incorporated bromodeoxyuridine and cell DNA content. C-myc mRNA expression was down-regulated and closely correlated to cell growth arrest. However, VDR expression in leukemic cell lines, as determined by immunofluorescence and Northern blot assays, was not consistently changed upon inhibition of cell proliferation since VDR levels were down-regulated only in HL-60 cells. Our data suggest that VDR expression cannot be explained simply as a reflection of the leukemic cell growth state.

Orthotopic tumorgrafts in nude mice as a model to evaluate calcitriol effects in breast cancer / Modelo de enxerto tumoral ortotópico em camundongos para avaliar os efeitos do calcitriol em câncer de mama

Abstract Calcitriol antiproliferative effects were observed in xenografts of breast cancer cell lines, however they were not yet investigated in tumorgrafts, consisting of freshly collected breast cancer samples xenografted into animals. Objectives To establish a tumorgraft model, from freshly collected breast cancer samples, which were directly implanted in nude mice, to study calcitriol effects. Methods Breast cancer samples collected from 12 patients were orthotopically implanted into nude mice. Animals were treated with weekly intratumoral injections of calcitriol 3 μg/Kg, which was previously shown to induce peak serum calcitriol levels in the predicted therapeutic range. Results Success engraftment rate was 25%. Tumorgrafts were established from aggressive (HER2 positive or histological grade 3) highly proliferative samples and original tumor characteristics were preserved. Calcitriol highly induced its target gene, CYP24A1, indicating that the genomic vitamin D pathway is active in tumorgrafts. However, no differences in the expression of proliferation and apoptosis markers (BrdU incorporation, Ki67, CDKN1A, CDKN1B, BCL2 expression) were observed in these highly proliferative tumor samples. Conclusions Tumorgrafts seem a promising model to explore other calcitriol doses and regimens, considering the heterogeneity of the disease and microenvironment interactions.

Resumo Os efeitos antiproliferativos de calcitriol foram observados em xenotransplantes de linhagens celulares de câncer de mama, entretanto, não foram ainda investigados em enxertos tumorais, consistindo de implantes em animais de amostras de câncer de mama recém-coletadas. Objetivos Estabelecer modelo de enxerto tumoral, a partir de amostra de câncer de mama recém-coletada e diretamente implantada em camundongos nude, para estudar o efeito do calcitriol. Métodos Amostras de câncer de mama de 12 pacientes foram implantadas ortotopicamente em camundongos nude. Os animais foram tratados com injeção intratumoral semanal de calcitriol 3 μg/Kg, a qual foi previamente associada com indução de pico sérico de calcitriol dentro do intervalo de nível terapêutico. Resultados A taxa de sucesso de pega do enxerto foi de 25%. Os enxertos tumorais foram estabelecidos de tumores agressivos com alta taxa de proliferação (HER2 positivo ou grau histológico 3) e as características do tumor original foram preservadas. O calcitriol induziu fortemente a expressão do gene alvo, CYP24A1, indicando que a via genômica da vitamina D está ativa nos enxertos tumorais, entretanto, não se observou diferenças na expressão de marcadores de proliferação e apoptose (incorporação de BrdU, expressão de Ki67, CDKN1A, CDKN1B e BCL2) nestas amostras altamente proliferativas. Conclusões Os enxertos tumorais parecem ser um modelo promissor para explorar outros esquemas e doses de calcitriol, considerando a heterogeneidade da doença e interações com o microambiente.