In vitro effects of two environmental toxicants, beauvericin and glyphosate in Roundup, on cell numbers and steroidogenesis of bovine ovarian cells.

Beauvericin is an emerging Fusariotoxin naturally occurring in cereal grains throughout the world whereas glyphosate (N-phosphonomethyl-glycine) is a non-selective systemic herbicide used worldwide. The purpose of this study is to evaluate a newly developed ovarian cell culture system (that includes both granulosa and theca cells) as an in vitro model for toxicological studies. Specifically, the effects of beauvericin and glyphosate in formulation with Roundup on ovarian cell numbers and steroid production were evaluated. Ovaries collected from cattle without luteal structures were sliced into 30-70 pieces each, and granulosa and theca cells were collected. Harvested cells were cultured for 48 h in 10% fetal bovine serum-containing medium followed by 48 h in serum-free medium containing testosterone (500 ng/mL; as an estrogen precursor) with the following eight treatments: (1) controls, (2) FSH (30 ng/mL) alone, (3) FSH plus insulin-like growth factor-1 (IGF1; 30 ng/mL), (4) FSH plus IGF1 plus beauvericin (3 µM), (5) FSH plus IGF1 plus glyphosate in Roundup (10 µg/mL), (6) FSH plus IGF1 plus fibroblast growth factor 9 (FGF9, 30 ng/mL), (7) a negative control without added testosterone, and (8) IGF1 plus LH (30 ng/mL) with basal medium without added testosterone. In the presence of FSH, IGF1 significantly increased cell numbers, estradiol and progesterone production by severalfold. Glyphosate in Roundup formulation significantly inhibited IGF1-induced cell numbers and estradiol and progesterone production by 89-94%. Beauvericin inhibited IGF1-induced cell numbers and estradiol and progesterone by 50-97% production. LH plus IGF1 significantly increased androstenedione secretion compared with controls without added testosterone indicating the presence of theca cells. In conclusion, the present study demonstrates that toxicological effects of beauvericin and glyphosate in Roundup formulation are observed in a newly developed ovarian cell model system and further confirms that both glyphosate and beauvericin may have the potential to impair reproductive function in cattle.

Gene-expression profiling of endomyocardial biopsies from dogs with dilated cardiomyopathy phenotype.

INTRODUCTION: The employment of advanced molecular biology technologies has expanded the diagnostic investigation of cardiomyopathies in dogs; these technologies have predominantly been performed on postmortem samples, although the recent use of endomyocardial biopsy in living dogs has enabled a better premortem diagnostic approach to study the myocardial injury. ANIMALS, MATERIALS, AND METHODS: Endomyocardial biopsies were collected in nine dogs with a dilated cardiomyopathy phenotype (DCM-p) and congestive heart failure and submitted to histologic examination, next-generation sequencing (NGS), and polymerase chain reaction analysis. Data from three healthy dogs (Fastq files) were retrieved from a previously approved study and used as a control group for ribonucleic acid sequencing. RESULTS: Histologic examination revealed endocardial fibrosis in six of nine dogs, whereas lymphocytic interstitial infiltrates were detected in two of nine dogs, and lymphoplasmacytic and macrophage infiltrates were detected in one of nine dogs. On polymerase chain reaction analysis, two dogs tested positive for canine parvovirus two and one dog for canine distemper virus. Gene-expression pathways involved in cellular energy metabolism (especially carbohydrates-insulin) and cardiac structural proteins were different in all DCM-p dogs compared to those in the control group. When dogs with lymphocytic interstitial infiltrates were compared to those in the control group, NGS analysis revealed the predominant role of genes related to inflammation and pathogen infection. CONCLUSIONS: Next-generation sequencing technology performed on in vivo endomyocardial biopsies has identified different molecular and genetic factors that could play a role in the development and/or progression of DCM-p in dogs.

Heart rhythm characterization during sudden cardiac death in dogs.

INTRODUCTION/OBJECTIVES: Inherited or acquired arrhythmic disorders and cardiac disease have been associated with sudden cardiac death (SCD) in dogs. The electrical mechanism related to death in most of these cases is unknown. This retrospective study aimed to describe arrhythmic events in dogs that experienced SCD during Holter monitoring. ANIMALS, MATERIALS AND METHODS: Nineteen client-owned dogs that experienced SCD during Holter examination were included. Clinical records from a Holter service database were reviewed, and both the rhythm preceding death and the dominant rhythm causing SCD were analysed. Clinical data, Holter diaries and echocardiographic diagnosis were also evaluated. RESULTS: Structural heart disease was identified in 12/19 dogs (dilated cardiomyopathy in five dogs, arrhythmogenic right ventricular cardiomyopathy in four dogs, myxomatous mitral valve disease in two dogs, and suspected myocarditis in one dog), five of which had concurrent congestive heart failure. Sudden cardiac death was related to ventricular premature complexes or monomorphic ventricular tachycardia degenerating into ventricular fibrillation in 42% of dogs, polymorphic ventricular tachycardia, or torsade de pointes-like inducing ventricular fibrillation in 21%, and asystole or presumptive agonal pulseless electrical activity triggered by malignant bradyarrhythmias in 37%. CONCLUSIONS: The most common rhythm associated with SCD in our population of dogs was ventricular tachycardia leading to ventricular fibrillation, although bradyarrhythmia-related SCD, possibly related to inappropriate vagal reflexes, was also a notable cause.

Radiofrequency catheter ablation of cranial vena cava flutter in four dogs.

Four dogs were referred to our institution for incessant supraventricular tachycardias causing weakness; congestive heart failure was present in one dog. At admission, all dogs had a surface electrocardiogram showing a narrow QRS complex tachycardia with a ventricular rate ranging from 80 to 300 bpm, variable atrioventricular conduction ratio from 1:1 to 3:1, and positive atrial depolarizations in inferior leads (II, II, III, and aVF), with isoelectric lines between them. Three of four dogs had a dilated cardiomyopathy phenotype; one dog had a heart base tumor involving the cranial vena cava wall. According to the electrocardiographic findings, a presumptive diagnosis of reverse typical or atypical atrial flutter was considered, and endocardial mapping was planned for each dog. During the electrophysiologic study, continuous atrial activation compatible with atypical atrial flutter was observed in all dogs, with concealed entrainment obtained at the level of the isthmus located at the distal portion of the cranial vena cava, close to the entrance into the right atrium. A linear radiofrequency catheter ablation was performed from the right atrial wall to the distal part of the cranial vena cava with a permanent interruption of the isthmic conduction in all dogs at a 6-month follow-up.

Effects of selected hormones and their combination on progesterone and estradiol production and proliferation of feline granulosa cells cultured in vitro.

Little is known about the hormonal regulation of feline ovarian granulosa cell proliferation and steroidogenesis. The present study aimed to develop a hormone responsive granulosa cell culture system to measure steroidogenic and cell proliferation responses to help identify factors that might regulate ovarian function in queens. Five experiments were conducted each with 75 or more ovaries, three in spring and two in fall seasons. Granulosa cells were isolated and treated in vitro with various hormones in serum-free medium for 48 h after an initial 48 h plating in 10% fetal calf serum. In granulosa cells isolated from spring and fall collected feline ovaries, IGF1 alone and combined with FSH stimulated (P < 0.05) cell proliferation, whereas FSH alone had no effect (P > 0.10) on cell proliferation. Also, in granulosa cells collected in spring and fall, IGF1 alone and FSH alone increased (P < 0.05) estradiol production by severalfold, and a combination of FSH and IGF1 increased (P < 0.05) estradiol production above either FSH or IGF1 treatment alone. The FSH plus IGF1 treatment increased (P < 0.05) CYP19A1 mRNA abundance by 27-fold. In contrast, EGF decreased (P < 0.05) FSH plus IGF1-induced estradiol production by over 80% in granulosa cells of both spring and fall collected ovaries. In granulosa cells isolated from spring and fall collected ovaries, IGF1 plus FSH inhibited (P < 0.05) progesterone production. Melatonin increased (P < 0.05) FSH plus IGF1-induced cell proliferation and amplified (P < 0.05) the FSH plus IGF1-induced inhibition of progesterone production. However, melatonin and GH had no effect (P > 0.10) on estradiol production either alone or in combination with FSH plus IGF1 in both spring and fall. Prolactin, FGF9 and activin had no effect (P > 0.10) on cell proliferation or steroidogenesis. FGF2 decreased (P < 0.05) estradiol production without affecting progesterone production or cell numbers. Growth differentiation factor 9 (GDF9) increased (P < 0.05) progesterone production but had no effect (P > 0.10) on granulosa cell proliferation or estradiol production. In conclusion, the in vitro system described herewithin may be useful to assess and evaluate ovarian function in feline species and has identified EGF, FSH and IGF1 as major regulators of feline ovarian follicular function.

Regulation of the transcription factor E2F1 mRNA in ovarian granulosa cells of cattle.

The E2F family of transcription factors plays an important role in the control of the cell cycle, cell proliferation, and differentiation, and their role in ovarian function is just emerging. Although some evidence suggests a possible role of E2F1 in ovarian follicular development, what regulates its production in ovarian cells is unknown. Objectives of this study were to determine whether: (i) E2F1 gene expression in granulosa cells (GCs) and theca cells (TCs) change with follicular development and (ii) E2F1 mRNA abundance in TC and GC is hormonally regulated. Using real-time PCR, E2F1 mRNA abundance in GC was 5.5-fold greater (P < 0.05) in small (SM; 1 to 5 mm) than large (LG; >8 mm) follicles, but in TC, E2F1 expression did not differ among follicle sizes. SM-follicle GC had 2.1-fold greater (P < 0.05) E2F1 mRNA than TC. In SM-follicle GC, FGF9 induced a 7.6-fold increase in E2F1 mRNA abundance; however, FGF9 did not affect (P > 0.10) abundance of E2F1 mRNA in LG-follicle TC or GC. Follicle-stimulating hormone (FSH) had no effect (P > 0.10) on E2F1 gene expression in SM- or LG-follicle GC. SM-follicle GC were concomitantly treated with insulin-like growth factor 1 (30 ng/mL), FSH (30 ng/mL), and either 0 or 30 ng/mL of FGF9 with or without 50 µM of an E2F inhibitor (E2Fi; HLM0064741); FGF9 alone increased (P < 0.05) GC numbers, whereas E2Fi alone decreased (P < 0.05) GC numbers, and concomitant treatment of E2Fi with FGF9 blocked (P < 0.05) this stimulatory effect of FGF9. Estradiol production was inhibited (P < 0.05) by FGF9 alone and concomitant treatment of E2Fi with FGF9 attenuated (P < 0.05) this inhibitory effect of FGF9. SM-follicle GC treated with E2Fi decreased (P < 0.05) E2F1 mRNA abundance by 70%. Collectively, our studies show that GC E2F1 mRNA is developmentally and hormonally regulated in cattle. Inhibition of E2F1 reduced FGF9-induced GC proliferation and attenuated FGF9-inhibited estradiol production, indicating that E2F1 may be involved in follicular development in cattle.

Effects of N-carbamylglutamate and arginine on steroidogenesis and proliferation of pig granulosa cells in vitro.

Results of in vivo studies indicate dietary N-carbamylglutamate (NCG) and arginine (ARG) can enhance reproductive performance in gilts. It was hypothesized that both NCG and ARG will alter hormone-induced estradiol (E2) production by granulosa cells (GC), explaining why these compounds could improve reproductive performance in pigs. The objective of these studies, therefore, was to evaluate the direct effects of NCG and ARG on porcine GC proliferation and steroidogenesis, using an in vitro cell culture system. The GC from small (SM; 1-5 mm) and large (LG; >5 mm) pig follicles were cultured for 2 days in 5% fetal bovine serum and 5% porcine serum-containing medium followed by 2 days in serum-free medium containing 500 ng/mL of testosterone (as an E2 precursor), and NCG or ARG at various doses in the presence of either follicle-stimulating hormone (FSH; 30 ng/mL), insulin-like growth factor-1 (IGF1; 30 ng/mL), or both. Numbers of GC were determined at the end of the experiment and concentrations of progesterone (P4) and E2 in culture medium were determined. Results indicated that LG-follicle GC were more responsive to NCG and ARG than SM-follicle GC. Specifically, in LG-follicle GC, NCG inhibited (P <  0.05) basal and FSH-induced P4 and E2 production but stimulated cell numbers; whereas ARG inhibited FSH-induced E2 production and cell numbers. In SM-follicle GC, treatment with NCG and ARG decreased IGF1 plus FSH induced P4 production, but E2 production and cell proliferation were not affected. These studies indicate that NCG and ARG may directly affect follicular function in pigs.

Regulation of the transcription factor E2F8 gene expression in bovine ovarian cells.

Overexpression of the transcription factor, E2F8, has been associated with ovarian cancer. Objectives of this study were to determine: 1) if E2F8 gene expression in granulosa cells (GC) and theca cells (TC) change with follicular development, and 2) if E2F8 mRNA abundance in TC and GC is hormonally regulated. Using real-time PCR, E2F8 mRNA abundance in GC and TC was greater (P < 0.05) in small than large follicles. FGF9 induced an increase (P < 0.05) in E2F8 mRNA abundance by 1.6- to 7-fold in large-follicle (8-20 mm) TC and GC as well as in small-follicle (1-5 mm) GC. Abundance of E2F8 mRNA in TC was increased (P < 0.05) with FGF2, FGF9 or VEGFA treatments alone in vitro, and concomitant treatment of VEGFA with FGF9 increased (P < 0.05) abundance of E2F8 mRNA above any of the singular treatments; BMP4, WNT3A and LH were without effect. IGF1 amplified the stimulatory effect of FGF9 on E2F8 mRNA abundance by 2.7-fold. Collectively, our studies show for the first time that follicular E2F8 is developmentally and hormonally regulated indicating that E2F8 may be involved in follicular development.

A slow-cycling subpopulation of melanoma cells with highly invasive properties.

Melanoma is a heterogeneous tumor with different subpopulations showing different proliferation rates. Slow-cycling cells were previously identified in melanoma, but not fully biologically characterized. Using the label-retention method, we identified a subpopulation of slow-cycling cells, defined as label-retaining cells (LRC), with strong invasive properties. We demonstrate through live imaging that LRC are leaving the primary tumor mass at a very early stage and disseminate to peripheral organs. Through global proteome analyses, we identified the secreted protein SerpinE2/protease nexin-1 as causative for the highly invasive potential of LRC in melanomas.

Holter monitoring in 36 dogs with myxomatous mitral valve disease.

OBJECTIVE: Describe the presence of arrhythmias in dogs with myxomatous mitral valve disease (MMVD) and the potential association with class of heart failure and left atrial enlargement. Compare the standard electrocardiogram (ECG) with Holter monitoring for assessing heart rate (HR). EXPERIMENTAL PROCEDURE: The study group of 36 dogs weighing less than 20 kg was divided into MMVD and no clinical signs (preclinical) or MMVD and clinical signs (clinical). A standard echocardiogram, ECG and 24-h Holter recording were obtained in all dogs. RESULTS: Minimum and mean Holter HRs were higher in the clinical group than in the preclinical group. Clinical dogs had more ventricular arrhythmias than preclinical dogs. An enlarged left atrium was associated with the presence of more supraventricular arrhythmias. CONCLUSIONS: Arrhythmias are a common finding in dogs with MMVD and Holter monitoring is a reliable tool for both HR monitoring and diagnosis.

Use of transcutaneous external pacing during transvenous pacemaker implantation in dogs.

The aim of this retrospective study was to evaluate the utility of transcutaneous external pacing (TEP) during transvenous pacemaker implantation in dogs. Eighty-two pacemakers were implanted in 77 dogs because of third-degree atrioventricular block (AVB) (58 cases; 70.7 per cent), sinus node dysfunction (SND) (nine cases; 11.0 per cent), high-grade second-degree AVB (six cases; 7.3 per cent), persistent atrial standstill (PAS) (four cases; 4.9 per cent), post-radiofrequency catheter ablation of the bundle of His (four cases; 4.9 per cent) and vasovagal syncope with atrial fibrillation (one case; 1.2 per cent). TEP was initiated during general anaesthesia after the onset of asystole or profound bradycardia, and stopped when permanent pacing was started. The use of TEP was necessary in 27 cases: 19 cases of third-degree AVB, five of SND, two of PAS and one of vasovagal syncope. External pacing was successful in all but two dogs.

Medición no invasiva de bilirrubina en el recién nacido. Significativo aporte a la práctica clínica en la ictericia neonatal / Non invasive measurement of serum bilirubin in newborns: significant contributions to the clinical practice in the jaundiced newborn

Objetivos del trabajo: 1) comparar la estimación de bilirrubina sérica obtenida por medición transcutánea, BiliCheck, con valores obtenidos espectrofotométricamente y por high performance liquid chromatography (Gold Standard), 2) Determinar puntos de corte de los valores de BiliCheck en los cuales se obtiene la máxima sensibilidad y especificidad, 3) Determinar si el color de la piel, peso de nacimiento, edad gestacional y sexo interfieren con la medición transcutánea de bilirrubina en recién nacidos

Enhancement of the efficacy of weekly low-dose taxotere by the long acting anti-prolactinemic drug cabergoline in pretreated metastatic breast cancer.

In view of its potential action as a growth factor, the evidence of abnormally high blood levels of prolactin (PRL) is associated with a poor prognosis in metastatic breast cancer. Moreover, metastatic breast cancer-related hyperprolactinemia has proven to counteract the efficacy of cancer chemotherapy. The negative influence of high blood levels of PRL on the efficacy of chemotherapy in metastatic breast cancer has been confirmed by previous preliminary studies, showing that the concomitant administration of the anti-prolactinemic dopaminergic agent bromocriptine may enhance the therapeutic effect of chemotherapy. However, the clinical use of bromocriptine is limited by its short duration and gastrointestinal toxicity. Therefore, new anti-prolactinemic drugs, characterized by less toxicity and a longer duration of activity, such as Cabergoline (CBG), could be more appropriated to control PRL secretion in breast cancer. On this basis, a study was planned to evaluate the efficacy and tolerability of a concomitant administration of CBG with weekly low-dose Taxotere (TXT) in pretreated metastatic breast cancer under chemotherapy. The study group comprised 70 metastatic breast cancer patients (females), pretreated with at least one previous chemotherapeutic line containing anthracyclines, who were randomized to be treated with TXT alone or TXT plus CBG. TXT 25 mg/m2 was given i.v. at weekly intervals for at least 9 consecutive cycles. CBG was given orally at 0.5 mg once per week. Abnormally high pre-treatment levels of PRL were seen in 24/70 (34%) patients, 11 of whom were treated with TXT plus CBG, whereas the other 13 received TXT alone. CBG induced a complete normalization of the PRL levels in all patients within the first two weeks of therapy, whereas no normalization of PRL occurred spontaneously in patients treated with chemotherapy alone. The objective tumor regression rate was significantly higher in patients concomitantly treated with CBG than in those who received chemotherapy alone (31/34 vs 13/36, p < 0.05), and this difference was particularly evident in patients with high PRL levels prior to therapy (6/11 vs 2/13). No CBG-related toxicity occurred. On the contrary, chemotherapy-induced asthenia was significantly lower in patients concomitantly treated with CBG (5/34 vs 11/36, p < 0.05). This study shows that the chemoneuroendocrine therapy of weekly low-dose TXT plus the anti-prolactinemic drug CBG is a new, effective and well-tolerated therapy for metastatic breast cancer. It may also be recommended in heavily pretreated patients or in those with poor clinical status.

Dissection of the functional and structural domains of phosphorelay histidine kinase A of Bacillus subtilis.

The initiation of sporulation in Bacillus subtilis results primarily from phosphoryl group input into the phosphorelay by histidine kinases, the major kinase being kinase A. Kinase A is active as a homodimer, the protomer of which consists of an approximately 400-amino-acid N-terminal putative signal-sensing region and a 200-amino-acid C-terminal autokinase. On the basis of sequence similarity, the N-terminal region may be subdivided into three PAS domains: A, B, and C, located from the N- to the C-terminal end. Proteolysis experiments and two-hybrid analyses indicated that dimerization of the N-terminal region is accomplished through the PAS-B/PAS-C region of the molecule, whereas the most amino-proximal PAS-A domain is not dimerized. N-terminal deletions generated with maltose binding fusion proteins showed that an intact PAS-A domain is very important for enzymatic activity. Amino acid substitution mutations in PAS-A as well as PAS-C affected the in vivo activity of kinase A, suggesting that both PAS domains are required for signal sensing. The C-terminal autokinase, when produced without the N-terminal region, was a dimer, probably because of the dimerization required for formation of the four-helix-bundle phosphotransferase domain. The truncated autokinase was virtually inactive in autophosphorylation with ATP, whereas phosphorylation of the histidine of the phosphotransfer domain by back reactions from Spo0F~P appeared normal. The phosphorylated autokinase lost the ability to transfer its phosphoryl group to ADP, however. The N-terminal region appears to be essential both for signal sensing and for maintaining the correct conformation of the autokinase component domains.

Abrogation of surgery-induced IL-6 hypersecretion by presurgical immunotherapy with IL-2 and its importance in the prevention of postoperative complications.

Because of its immunosuppressive effect, surgery-induced immunosuppression may depend at least in part on the postoperative hypersecretion of IL-6, which is also responsible for surgical complications. Most of the immunosuppressive events induced by surgery, including lymphocytopenia, NK and T lymphocyte decline, and dendritic cell deficiency have been proven to be abrogated by a preoperative injection of IL-2 for few days. However, the cytokine mechanisms responsible for IL-2-induced abrogation of surgery-related immunosuppression need to be better investigated and understood. This study was performed to analyze the influence of IL-2 presurgical immunotherapy on IL-6 secretion in the postoperative period. The study was performed in 12 operable colorectal cancer patients, who were preoperatively pretreated with IL-2 (12 million lU/day subcutaneously for 3 consecutive days before surgery). The control group consisted of 21 age-and disease-matched colorectal cancer patients who underwent surgery without a preoperative immunotherapy with IL-2. Serum levels of IL-6 were measured by an enzyme immunoassay before surgery, and at days 3 and 7 of the postoperative period. A significant increase in mean serum levels of IL-6 occurred in the postoperative period only in the control patients, whereas in the IL-2 pretreated group no significant difference was seen between presurgical and postoperative IL-6 mean concentrations. The difference between controls and IL-2 group was particularly evident for patients with abnormally elevated presurgical values of IL-6. This study, by showing that a presurgical injection of IL-2 may prevent surgery-induced IL-6 enhanced secretion, would suggest that the previously described neutralization of surgery-induced immunosuppression by IL-2 preoperative immunotherapy may depend at least in part on the inhibition of postoperative production of IL-6, whose immunosuppressive effects have been well demonstrated at least on anticancer immunity.

[Variations in interleukin-2 and interleukin-6 due to surgical trauma in cancer patients]. / Variazioni di interleuchina-2 e interleuchina-6 da trauma chirurgico in pazienti oncologici.

Inflammatory response after surgical trauma, which is necessary for infection control and tissue repairing, can actually produce some cytokines suppressive of the antitumoral immunity response. In this study the authors evaluate pre- and post-operative IL-2 (antitumor response activator) and IL-6 (lymphocytic response inhibitor and tumor growth factor) levels in 26 cancer patients undergoing resective surgery. Analysis of the results showed a significative IL-6 increase and a tendency to IL-2 decrease in the post-operative period. It is thus confirmed, even on the basis of the cytokines, the meaningful immunosuppressive effect of the surgical trauma on neoplastic growth control.

[Gastrointestinal stromal tumor as the cause of intestinal hemorrhage: description of a clinical case]. / Tumore stromale gastrointestinale come causa di enterorragia: descrizione di un caso clinico.

The term gastrointestinal stromal tumor describes a heterogeneous group of tumors of mesenchymal origin with particular histologic features. Their classification has recently been made possible thanks to numerous immunohistochemical and ultrastructural studies. We report the case of a patient who came to our attention because of serious anemia due to a gastrointestinal stromal tumor located between the second and third portion of the duodenum. This pathology, although not frequent, should be considered in the differential diagnosis of gastrointestinal bleeding. In these cases it is important to perform careful endoscopic inspection to the third portion of th duodenum, even when previous tracts evidence lesions that could be responsible for the patient's symptoms.

Rapid capillary zone electrophoresis in isoelectric histidine buffer: high resolution of the poly-T tract allelic variants in intron 8 of the CFTR gene.

The poly-T tract in intron 8 of the cystic fibrosis conductance transmembrane regulator (CFTR) gene exists in three variants, 5T, 7T, and 9T. The 7T and 9T variants generate a predominantly normal transcript, whereas the 5T variant engenders an anomalous product. The analysis of the poly-T tract is assuming increasing relevance, both to assess the implication of the CFTR gene in congenital bilateral absence of the vas deferens and to evaluate genotype-phenotype correlation in cystic fibrosis. Mapping of the poly-T tract has been performed by cumbersome and time-consuming methodologies. Capillary zone electrophoresis, combined with laser-induced fluorescence detection, was introduced for a rapid separation of the poly-T tract amplified products. As separation buffer, we adopted 200 mmol/L histidine (pH = pI = 7.6), and the capillary was filled with 10% polyacrylamide, allowing separations in less than 10 min. Capillary zone electrophoresis results were in perfect agreement with dot-blot analysis.

Internal mammary blood supply for ileo-colon interposition in esophagogastroplasty: a case report.

We report on a clinical case where microsurgical techniques successfully supported traditional surgery in a wide reconstruction between the oropharynx and small bowel. Several years ago, the patient sustained a severe corrosive injury of the upper digestive tract with subsequent esophageal stricture and stiffening; at that time, an emergency gastrectomy was performed. In this case, the restoration of the defect could not rely on the classic colonic interposition. During the operation the ileo-colic flap, well-fitted for tension-free reconstruction, revealed the foreseen inadequacy of its vascularization based on the sole middle colic vascular pedicle. The blood supply to its proximal part was then increased by microanastomosis between the right internal mammary and ileo-colic vessels. The revascularization ensured the viability of the interposed tissue. Oral intake resumed after 3 weeks; nowadays the patient is able to maintain her ideal weight with adequate nutrition.

Interleukin-12 in early or advanced cancer patients.

Interleukin-2 (IL-2) and interleukin-12 (IL-12) may represent the most important antitumour cytokines in human neoplasms. IL-2 blood levels decrease in advanced solid malignancies, but currently there are no data on IL-12 secretion in cancer patients. This study was performed to obtain preliminary data about IL-12 secretion in patients with solid malignant tumours, either in relation to the extension of disease, or to other cytokines, including IL-2, IL-6 and IL-10. The study included 40 solid cancer patients, 24 of whom showed distant organ metastases. Cytokine serum levels were measured by an enzyme immunoassay of blood samples collected during the morning. No patient had abnormally low levels of IL-12, but the levels were high in 14/14 (35%) patients. Mean levels of IL-12 were significantly higher in metastatic patients compared with non-metastatic patients (P < 0.05). Moreover, metastatic patients with high blood concentrations of IL-12 showed significantly lower levels of IL-10 than metastatic patients with normal IL-12 values, while no difference was seen in IL-2 mean concentrations. IL-6 mean levels were lower in metastatic patients with increased IL-12 levels, but this was non-significant. This preliminary study shows that advanced solid cancers are not characterised by a diminished secretion of IL-12, but rather IL-12 levels tend to be abnormally high in metastatic cancer patients.