Human Dermal Decellularized ECM Hydrogels as Scaffolds for 3D In Vitro Skin Aging Models.

Biomaterials play an important role in the development of advancing three dimensional (3D) in vitro skin models, providing valuable insights for drug testing and tissue-specific modeling. Commercial materials, such as collagen, fibrin or alginate, have been widely used in skin modeling. However, they do not adequately represent the molecular complexity of skin components. On this regard, the development of novel biomaterials that represent the complexity of tissues is becoming more important in the design of advanced models. In this study, we have obtained aged human decellularized dermal extracellular matrix (dECM) hydrogels extracted from cadaveric human skin and demonstrated their potential as scaffold for advanced skin models. These dECM hydrogels effectively reproduce the complex fibrillar structure of other common scaffolds, exhibiting similar mechanical properties, while preserving the molecular composition of the native dermis. It is worth noting that fibroblasts embedded within human dECM hydrogels exhibit a behavior more representative of natural skin compared to commercial collagen hydrogels, where uncontrolled cell proliferation leads to material shrinkage. The described human dECM hydrogel is able to be used as scaffold for dermal fibroblasts in a skin aging-on-a-chip model. These results demonstrate that dECM hydrogels preserve essential components of the native human dermis making them a suitable option for the development of 3D skin aging models that accurately represent the cellular microenvironment, improving existing in vitro skin models and allowing for more reliable results in dermatopathological studies.

First case report of M1 macrophage polarization in an untreated symptomatic patient with toxoplasmosis.

BACKGROUND: In immunocompetent patients, acute toxoplasmosis is usually asymptomatic. We identified M1 macrophages in a case of symptomatic acute Toxoplasma gondii infection that resolved without treatment. M1 macrophages have been demonstrated in animal models of toxoplasmosis, but not in humans. CASE PRESENTATION: A 63-year-old woman presented with laterocervical and axillary bilateral lymphadenopathy. Her anamnesis defined an episode of high fever and prolonged asthenia 4 months previously, which suggested an infectious disease. Following laboratory, radiological, and pathological analyses, she was diagnosed with toxoplasmosis. Immunohistochemical analyses were performed on lymph node sections. More than 50% of the macrophages in the lymph node microgranulomas were M1 macrophages, defined by CD68+/p-Stat1+ staining, and the presence of T helper 1 lymphocytes indicated an immune response known to induce M1 macrophage polarization. Activated endothelial cells were found only in inflamed areas. No therapy was administered before or after diagnosis, and the lymphadenopathy resolved after a follow-up of 5 months. CONCLUSIONS: This is the first report to demonstrate the presence of M1 macrophages in human toxoplasmosis. Our findings contribute to the understanding of the pathogenesis of toxoplasmosis, and encourage further studies on the role of macrophage polarization in human toxoplasmosis.

Effects of Fe deficiency on the protein profiles and lignin composition of stem tissues from Medicago truncatula in absence or presence of calcium carbonate.

UNLABELLED: Iron deficiency is a yield-limiting factor with major implications for crop production, especially in soils with high CaCO3. Because stems are essential for the delivery of nutrients to the shoots, the aim of this work was to study the effects of Fe deficiency on the stem proteome of Medicago truncatula. Two-dimensional electrophoresis separation of stem protein extracts resolved 276 consistent spots in the whole experiment. Iron deficiency in absence or presence of CaCO3 caused significant changes in relative abundance in 10 and 31 spots, respectively, and 80% of them were identified by mass spectrometry. Overall results indicate that Fe deficiency by itself has a mild effect on the stem proteome, whereas Fe deficiency in the presence of CaCO3 has a stronger impact and causes changes in a larger number of proteins, including increases in stress and protein metabolism related proteins not observed in the absence of CaCO3. Both treatments resulted in increases in cell wall related proteins, which were more intense in the presence of CaCO3. The increases induced by Fe-deficiency in the lignin per protein ratio and changes in the lignin monomer composition, assessed by pyrolysis-gas chromatography-mass spectrometry and microscopy, respectively, further support the existence of cell wall alterations. BIOLOGICAL SIGNIFICANCE: In spite of being essential for the delivery of nutrients to the shoots, our knowledge of stem responses to nutrient deficiencies is very limited. The present work applies 2-DE techniques to unravel the response of this understudied tissue to Fe deficiency. Proteomics data, complemented with mineral, lignin and microscopy analyses, indicate that stems respond to Fe deficiency by increasing stress and defense related proteins, probably in response of mineral and osmotic unbalances, and eliciting significant changes in cell wall composition. The changes observed are likely to ultimately affect solute transport and distribution to the leaves.

Effects of Fe deficiency on the protein profile of Brassica napus phloem sap.

The aim of this work was to study the effect of Fe deficiency on the protein profile of phloem sap exudates from Brassica napus using 2DE (IEF-SDS-PAGE). The experiment was repeated thrice and two technical replicates per treatment were done. Phloem sap purity was assessed by measuring sugar concentrations. Two hundred sixty-three spots were consistently detected and 15.6% (41) of them showed significant changes in relative abundance (22 decreasing and 19 increasing) as a result of Fe deficiency. Among them, 85% (35 spots), were unambiguously identified. Functional categories containing the largest number of protein species showing changes as a consequence of Fe deficiency were signaling and regulation (32%), and stress and redox homeostasis (17%). The Phloem sap showed a higher oxidative stress and significant changes in the hormonal profile as a result of Fe deficiency. Results indicate that Fe deficiency elicits major changes in signaling pathways involving Ca and hormones, which are generally associated with flowering and developmental processes, causes an alteration in ROS homeostasis processes, and induces decreases in the abundances of proteins involved in sieve element repair, suggesting that Fe-deficient plants may have an impaired capacity to heal sieve elements upon injury.

Protein profile of Beta vulgaris leaf apoplastic fluid and changes induced by Fe deficiency and Fe resupply.

The fluid collected by direct leaf centrifugation has been used to study the proteome of the sugar beet apoplastic fluid as well as the changes induced by Fe deficiency and Fe resupply to Fe-deficient plants in the protein profile. Plants were grown in Fe-sufficient and Fe-deficient conditions, and Fe resupply was carried out with 45 µM Fe(III)-EDTA for 24 h. Protein extracts of leaf apoplastic fluid were analyzed by two-dimensional isoelectric focusing-SDS-PAGE electrophoresis. Gel image analysis revealed 203 consistent spots, and proteins in 81% of them (164) were identified by nLC-MS/MS using a custom made reference repository of beet protein sequences. When redundant UniProt entries were deleted, a non-redundant leaf apoplastic proteome consisting of 109 proteins was obtained. TargetP and SecretomeP algorithms predicted that 63% of them were secretory proteins. Functional classification of the non-redundant proteins indicated that stress and defense, protein metabolism, cell wall and C metabolism accounted for approximately 75% of the identified proteome. The effects of Fe-deficiency on the leaf apoplast proteome were limited, with only five spots (2.5%) changing in relative abundance, thus suggesting that protein homeostasis in the leaf apoplast fluid is well-maintained upon Fe shortage. The identification of three chitinase isoforms among proteins increasing in relative abundance with Fe-deficiency suggests that one of the few effects of Fe deficiency in the leaf apoplast proteome includes cell wall modifications. Iron resupply to Fe deficient plants changed the relative abundance of 16 spots when compared to either Fe-sufficient or Fe-deficient samples. Proteins identified in these spots can be broadly classified as those responding to Fe-resupply, which included defense and cell wall related proteins, and non-responsive, which are mainly protein metabolism related proteins and whose changes in relative abundance followed the same trend as with Fe-deficiency.

Role of E6/E7 mRNA test in the diagnostic algorithm of HPV-positive patients showing ASCUS and LSIL: clinical and economic implications in a publicly financed healthcare system.

OBJECTIVE: Colposcopy is widely used to triage women with mild cervical abnormalities. However, this approach is associated with low specificity and predictive value. The efficacy of E6/E7 mRNA test for this purpose has been demonstrated, but studies estimating its cost-effectiveness are still lacking. Given the limited healthcare financial resources, such an evaluation is a priority. METHODS: We analyzed the clinical history of 432 women referred to colposcopy and colposcopy-directed biopsy for persisting ASCUS and LSIL, and compared three alternative triage protocols: immediate colposcopy; reflex HPV DNA testing and HPV DNA plus mRNA tests in sequence. RESULTS: Molecular tests in sequence significantly reduce colposcopy referral, cost for assessed women, and cost for CIN2 detected. On the other hand, incremental cost-effectiveness ratio of this protocol was the highest. CONCLUSION: Our preliminary data, providing an estimation of the economic burden deriving from the introduction of E6/E7 mRNA test in the triage algorithm of patients with mild cervical abnormalities, may be useful for future healthcare policy.

The distinct functional roles of the inner and outer chloroplast envelope of Pea (Pisum sativum) as revealed by proteomic approaches.

Protein profiles of inner (IE) and outer (OE) chloroplast envelope membrane preparations from pea were studied using shotgun nLC-MS/MS and two-dimensional electrophoresis, and 589 protein species (NCBI entries) were identified. The relative enrichment of each protein in the IE/OE pair of membranes was used to provide an integrated picture of the chloroplast envelope. From the 546 proteins identified with shotgun, 321 showed a significant differential distribution, with 180 being enriched in IE and 141 in OE. To avoid redundancy and facilitate in silico localization, Arabidopsis homologues were used to obtain a nonredundant list of 409 envelope proteins, with many showing significant OE or IE enrichment. Functional classification reveals that IE is a selective barrier for transport of many metabolites and plays a major role in controlling protein homeostasis, whereas proteins in OE are more heterogeneous and participate in a wide range of processes. Data support that metabolic processes previously described to occur in the envelope such as chlorophyll and tocopherol biosynthesis can be ascribed to the IE, whereas others such as carotenoid or lipid biosynthesis occur in both membranes. Furthermore, results allow empirical assignation to the IE and/or OE of many proteins previously assigned to the bulk chloroplast envelope proteome.

A very rare case of HPV-53-related cervical cancer, in a 79-year-old woman with a previous history of negative Pap cytology.

The introduction of organized cervical cancer (CC) screening programs has drastically reduced the prevalence of CC. However the incidence is still too high, especially among elderly women. All guidelines strongly recommend a regular Papanicolaou (Pap) testing for young and middle-aged patients. On the other hand, many international professional societies no longer advise screening in women who have undergone hysterectomy, and in women aged 65 years and above, who have a previous history of regular Pap smears. Here we report the case of poorly differentiated CC, involving the pelvic lymph nodes and urinary bladder, occurring in a 79-year-old woman who regularly underwent Pap tests, with no reported cytological abnormalities. In this very rare case, the CC cells, as well as cells from metastatic lymph nodes and cells from urinary specimens, molecularly showed human papilloma virus (HPV)-53. With the limitations of a single case, this report brings important information to prevent CC in elderly patients: the utility of molecular tests to increase sensitivity of Pap smears in postmenopausal women; the importance of HPV-53 as one of the four "emergent" genotypes having a possible role in oncogenesis; and the presence of HPV-53 in lymph node metastases from cervical carcinoma, which would support the role of this virus in the maintenance of malignant status.

Chromogenic in situ hybridization and p16/Ki67 dual staining on formalin-fixed paraffin-embedded cervical specimens: correlation with HPV-DNA test, E6/E7 mRNA test, and potential clinical applications.

Although HPV-DNA test and E6/E7 mRNA analyses remain the current standard for the confirmation of human papillomavirus (HPV) infections in cytological specimens, no universally adopted techniques exist for the detection of HPV in formalin-fixed paraffin-embedded samples. Particularly, in routine laboratories, molecular assays are still time-consuming and would require a high level of expertise. In this study, we investigated the possible use of a novel HPV tyramide-based chromogenic in situ hybridization (CISH) technology to locate HPV on tissue specimens. Then, we evaluate the potential usefulness of p16(INK4a)/Ki-67 double stain on histological samples, to identify cervical cells expressing HPV E6/E7 oncogenes. In our series, CISH showed a clear signal in 95.2% of the specimens and reached a sensitivity of 86.5%. CISH positivity always matched with HPV-DNA positivity, while 100% of cases with punctated signal joined with cervical intraepithelial neoplasia grade 2 or worse (CIN2+). p16/Ki67 immunohistochemistry gave an interpretable result in 100% of the cases. The use of dual stain significantly increased the agreement between pathologists, which reached 100%. Concordance between dual stain and E6/E7 mRNA test was 89%. In our series, both CISH and p16(INK4a)/Ki67 dual stain demonstrated high grade of performances. In particular, CISH would help to distinguish episomal from integrated HPV, in order to allow conclusions regarding the prognosis of the lesion, while p16(INK4a)/Ki67 dual stain approach would confer a high level of standardization to the diagnostic procedure.

Changes induced by zinc toxicity in the 2-DE protein profile of sugar beet roots.

Changes induced by three levels of Zn toxicity in the root proteome from Beta vulgaris were studied by two dimensional gel electrophoresis. 320 spots were consistently detected and 5, 5 and 11% of them showed significant changes in relative abundance as a result of the 50, 100 and 300µM Zn treatments, respectively, when compared to controls (1.2µM Zn). Forty-four spots had consistent changes between all treatments, and 93% were identified. At low and mild Zn excess, the complex I of the mitochondrial transport chain and the oxidative phosphorylation were mildly impaired, and an effort to compensate this effect by increasing glycolysis was observed. At high Zn excess, a general metabolism shutdown occurred, as denoted by decreases in the aerobic respiration and by an impairment of the defense systems against oxidative stress. Accordingly, lipid peroxidation increased as Zn supply increased. This study suggests that metabolic changes at high Zn supply reflect cell death, while changes at low and mild Zn supplies may rather explain the metabolic reprogramming occurring upon Zn toxicity. Results also suggest that Zn competition with divalent ions including Fe may contribute to many of the Zn toxicity symptoms, especially at low and moderate Zn supplies. BIOLOGICAL SIGNIFICANCE: Results in this work provide a comprehensive overview of the effects of Zn toxicity in roots of sugar beet plants. Effects at low and mild Zn excess are similar and reflect changes in the metabolism aimed to overcome this heavy metal stress, whereas effects at high Zn supply indicate a general shutdown of the metabolism and cell death. Our results indicate that Zn toxicity elicits major impairments in the oxidative stress defense systems, possibly due to Zn competition with divalent cations including Fe, in spite that Zn is not a redox active element by itself.

Protein profile of Lupinus texensis phloem sap exudates: searching for Fe- and Zn-containing proteins.

The aim of this study was to obtain a comprehensive overview of the phloem sap protein profile of Lupinus texensis, with a special focus on proteins binding Fe and Zn. L. texensis was chosen as model plant given the simplicity to obtain exudates from sieve elements. Protein profiling by 2DE revealed 249 spots, and 54 of them were unambiguously identified by MALDI-MS and ESI-MS/MS. The largest number of identified protein species belongs to protein modification/turnover and general metabolism (19-21%), followed by redox homeostasis (9%) and defense and cell structural components (7%). This protein profile is similar to that reported in other plant species, suggesting that the phloem sap proteome is quite conserved. Staining of 2DE gels for Fe-containing proteins and affinity chromatography experiments revealed the presence of two low molecular weight Fe-binding proteins in phloem sap: a metallothionein-like protein type 2B identified in the Fe-affinity chromatography, and a second protein identified with both Fe staining methods. This protein species had a molecular weight of 13.5 kDa, a pI of 5.6 and 51% homology to a phloem-specific protein from Medicago truncatula. Zinc affinity chromatography revealed four Zn-binding proteins in phloem sap, one belonging to the dehydrin family and three Zn finger proteins.

Iron-dependent modifications of the flower transcriptome, proteome, metabolome, and hormonal content in an Arabidopsis ferritin mutant.

Iron homeostasis is an important process for flower development and plant fertility. The role of plastids in these processes has been shown to be essential. To document the relationships between plastid iron homeostasis and flower biology further, a global study (transcriptome, proteome, metabolome, and hormone analysis) was performed of Arabidopsis flowers from wild-type and triple atfer1-3-4 ferritin mutant plants grown under iron-sufficient or excess conditions. Some major modifications in specific functional categories were consistently observed at these three omic levels, although no significant overlaps of specific transcripts and proteins were detected. These modifications concerned redox reactions and oxidative stress, as well as amino acid and protein catabolism, this latter point being exemplified by an almost 10-fold increase in urea concentration of atfer1-3-4 flowers from plants grown under iron excess conditions. The mutant background caused alterations in Fe-haem redox proteins located in membranes and in hormone-responsive proteins. Specific effects of excess Fe in the mutant included further changes in these categories, supporting the idea that the mutant is facing a more intense Fe/redox stress than the wild type. The mutation and/or excess Fe had a strong impact at the membrane level, as denoted by the changes in the transporter and lipid metabolism categories. In spite of the large number of genes and proteins responsive to hormones found to be regulated in this study, changes in the hormonal balance were restricted to cytokinins, especially in the mutant plants grown under Fe excess conditions.

Changes induced by Fe deficiency and Fe resupply in the root protein profile of a peach-almond hybrid rootstock.

The changes in the root extract protein profile of the Prunus hybrid GF 677 rootstock (P. dulcis × P. persica) grown in hydroponics as affected by Fe deficiency and short-term (24 h) Fe resupply have been studied by 2-dimensional gel electrophoresis-based techniques. A total of 335 spots were consistently found in the gels. Iron deficiency caused above 2-fold increases or >50% decreases in the relative abundance in 10 and 6 spots, respectively, whereas one spot was only detected in Fe-deficient plants. Iron resupply to Fe-deficient plants caused increases and decreases in relative abundance in 15 and 16 spots, respectively, and one more spot was only detected in Fe-resupplied Fe-deficient plants. Ninety-five percent of the proteins changing in relative abundance were identified using nanoliquid chromatography-tandem mass spectrometry. Defense responses against oxidative and general stress accounted for 50% of the changes in Fe-deficient roots. Also, a slight induction of the glycolysis-fermentation pathways was observed in GF 677 roots with Fe deficiency. The root protein profile of 24 h Fe-resupplied plants was similar to that of Fe-deficient plants, indicating that the deactivation of Fe-deficiency metabolic responses is slow. Taken together, our results suggest that the high tolerance of GF 677 rootstock to Fe deficiency may be related to its ability to elicit a sound defense response against both general and oxidative stress.

Intravenous pyogenic granuloma of the right adrenal gland: report of a case.

Pyogenic granuloma (PG) is a tumor-like lesion that typically arises on human skin. Intravenous pyogenic granuloma (IVPG) is the vascular counterpart, mostly observed in the venous structures of the neck and upper extremities. Chronic irritation of the skin, traumatic injury, and hormonal alterations seem to be implicated in the pathogenesis of PG. The incidence of PG, and IVPG, is very low in the reported scientific literature, and this underlines the need for understanding unresolved questions concerning the uncommon presentation, and correct diagnosis before surgical intervention. This report describes a case of IVPG diagnosed in a 55-year-old female that presented for observation of chronic abdominal pain associated with nausea and anorexia. A well-defined mass located in the right adrenal gland was documented by ultrasonography and finally confirmed by contrast enhanced CT of the abdomen. There were no radiological signs of liver, kidney, or vascular infiltration. The 35 × 22 mm adrenal gland lesion, suspected to be an adrenal gland tumor, was resected using a minimally invasive approach. Laparoscopic right adrenalectomy was successfully performed. The histology documented the typical morphological features of IVPG in the context of a normal right adrenal gland. This report describes and discusses the unusual presentation of intraabdominal IVPG located in the adrenal gland region together with a review of the current literature.

A diagnostic dilemma: dystrophic calcified nodule of the testicle in a patient with no other symptoms - case report and review of the literature.

Dystrophic calcified nodule of the testicle represents an exceptional lesion with an unknown etiology and controversial diagnostic approach. There are very few reports in the literature that have examined this feature. Here, we report the fourth case of dystrophic testis nodule, identified in a 46-year-old man who presented the urologist with a palpable mass on his left testicle and no other symptoms. Histopathological findings were discussed and pooled with those of all previously published series. We believe that this description is particularly innovative because of the absence of symptoms and testicular pain associated with the lesion, and interesting as an attempt to determine the recognition of this rare entity.

Spatially resolved analysis of small molecules by matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI).

• Matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) of tissues provides the means to analyse the spatial distributions of small molecules and proteins within tissues. This imaging technique is commonplace in medicinal and pharmaceutical research, but its application in plant science is very recent. Broader introduction requires specific adaptations for plant tissues. Sample preparation is of paramount importance in order to obtain high-quality spectra providing sufficient spatial resolution for compounds. Optimization is required for sectioning, choice of matrix and means of matrix deposition. • Here, we present our current protocols for the detection of small molecules in cryodissected immature barley (Hordeum vulgare) grains and tobacco (Nicotiana tabacum) roots. • Examples of MALDI-MSI measurements are provided, and the level of reproducibility across biological replicates is addressed. Furthermore, our approaches for the validation of distribution patterns and for the identification of molecules are described. • Finally, we discuss how MALDI-MSI can contribute to applied plant research.

Root responses of Medicago truncatula plants grown in two different iron deficiency conditions: changes in root protein profile and riboflavin biosynthesis.

Iron deficiency is a yield-limiting factor with major implications for field crop production in one-third of the world's agricultural areas, especially those with high soil CaCO(3). In the present work, a two-dimensional gel electrophoresis proteomic approach was combined with a study on the riboflavin synthesis pathway, including qPCR and riboflavin determination, to investigate Fe-deficiency responses in Medicago truncatula plants grown with and without CaCO(3). Iron deficiency caused a de novo accumulation of DMRLs and GTPcII, proteins involved in riboflavin biosynthesis, as well as marked increases in root riboflavin concentrations and in the expression of four genes from the riboflavin biosynthetic pathway. Two novel changes found were the increased accumulation of proteins related to N recycling and protein catabolism. Other identified changes were consistent with previously found increases in glycolysis, TCA cycle, and stress-related processes. All effects were more marked in the presence of CaCO(3). Our results show that the riboflavin biosynthesis pathway was up-regulated at the genomic, proteomic, and metabolomic levels under both Fe-deficiency treatments, especially in the presence of CaCO(3). Results also indicate that N recycling occurs in M. truncatula upon Fe deficiency, possibly constituting an additional anaplerotic N and C source for the synthesis of secondary metabolites, carboxylates, and others.

An in vivo model of Met-driven lymphoma as a tool to explore the therapeutic potential of Met inhibitors.

PURPOSE: Met, the tyrosine kinase receptor for hepatocyte growth factor, is frequently deregulated in human cancer. Recent evidence indicates that Met amplification may confer resistance to treatments directed toward other receptor tyrosine kinases. Thus, there is a need to develop Met inhibitors into therapeutic tools, to be used alone or in combination with other molecularly targeted drugs. Preclinical validation of Met inhibitors has thus far been done in nude mice bearing cancer cells xenografts. A far superior model would be a transgenic line developing spontaneous Met-driven tumors with high penetrance and short latency. EXPERIMENTAL DESIGN: To this end, we introduced into the mouse genome TPR-MET, the oncogenic form of MET. The Tpr-Met protein ensures deregulation of Met signaling because dimerization motifs in the Tpr moiety cause ligand-independent activation of the Met kinase. RESULTS: Here, we describe a TPR-MET transgenic line that develops thymic T-cell lymphoma with full penetrance and very short latency. In the tumors, Tpr-Met and its effectors were phosphorylated. Treatment of tumor-derived T lymphocytes with the selective Met inhibitor PHA-665752 at nanomolar concentrations abolished phosphorylation of Met and downstream effectors and led to caspase-mediated apoptosis. I.v. administration of PHA-665752 to transgenic mice bearing lymphomas in exponential growth phase led to a significant decrease in tumor growth and, in some cases, to tumor regression. CONCLUSIONS: Our transgenic line, which within 2 months reliably develops Tpr-Met-driven T-cell lymphoma, represents a valuable tool to explore the efficacy and therapeutic potential of Met kinase inhibitors as anticancer drugs.

Chemiluminescence determination of sulphadiazine in drugs by flow injection analysis using the peroxyoxalate reaction in micellar medium.

Peroxyoxalate chemiluminescence (PO-CL) is an indirect type of CL which allows the detection of native fluorophores or compounds derivatized with fluorescent labels. We propose a flow injection analysis (FIA) configuration based on the use of a two-injection valve system for the introduction of both PO and derivatized analyte solutions in the flow system, avoiding the problems arising from the use of organic solvents, such as acetonitrile, as no special tubes nor special pumps are required. Furthermore, the use of micellar media (sodium dodecyl sulphate, SDS) as a carrier and the addition of tetrahydrofurane (THF) in the PO solutions increase both the solubility and stability of POs, avoiding their rapid degradation in water. The proposed CL-FIA system has been applied to the determination of sulphadiazine (a sulphonamide mainly used in the treatment of urinary tract infections for human and veterinary use) using bis[2,4,6-trichlorophenyl]oxalate (TCPO) as CL precursor, H2O2 as oxidant, imidazole as a catalyst and fluorescamine as the fluorescent derivatizing agent. The optimization of variables was carried out by means of experimental designs and the method showed a LOQ of 379 microg l(-1) (calibration range 126-2000 microg l(-1)). It has been satisfactorily applied to the quantification of sulphadiazine in pills for human use and ampoules for veterinary use.