BDNF and AMPA receptors in the cNTS modulate the hyperglycemic reflex after local carotid body NaCN stimulation.

The application of sodium cyanide (NaCN) to the carotid body receptors (CBR) (CBR stimulation) induces rapid blood hyperglycemia and an increase in brain glucose retention. The commissural nucleus tractus solitarius (cNTS) is an essential relay nucleus in this hyperglycemic reflex; it receives glutamatergic afferents (that also release brain derived neurotrophic factor, BDNF) from the nodose-petrosal ganglia that relays CBR information. Previous work showed that AMPA in NTS blocks hyperglycemia and brain glucose retention after CBR stimulation. In contrast, BDNF, which attenuates glutamatergic AMPA currents in NTS, enhances these glycemic responses. Here we investigated the combined effects of BDNF and AMPA (and their antagonists) in NTS on the glycemic responses to CBR stimulation. Microinjections of BDNF plus AMPA into the cNTS before CBR stimulation in anesthetized rats, induced blood hyperglycemia and an increase in brain arteriovenous (a-v) of blood glucose concentration difference, which we infer is due to increased brain glucose retention. By contrast, the microinjection of the TrkB antagonist K252a plus AMPA abolished the glycemic responses to CBR stimulation similar to what is observed after AMPA pretreatments. In BDNF plus AMPA microinjections preceding CBR stimulation, the number of c-fos immunoreactive cNTS neurons increased. In contrast, in the rats microinjected with K252a plus AMPA in NTS, before CBR stimulation, c-fos expression in cNTS decreased. The expression of AMPA receptors GluR2/3 did not change in any of the studied groups. These results indicate that BDNF in cNTS plays a key role in the modulation of the hyperglycemic reflex initiated by CBR stimulation.

Nitric oxide in the solitary tract nucleus (STn) modulates glucose homeostasis and FOS-ir expression after carotid chemoreceptor stimulation.

We evaluate in rats the role of NO in the solitary tract nucleus (STn) after an anoxic stimulus to carotid body chemoreceptor cells (CChrc) with cyanide (NaCN), on the hyperglycemic reflex with glucose retention by the brain (BGR) and FOS expression (FOS-ir) in the STn. The results suggest that nitroxidergic pathways in the STn may play an important role in glucose homeostasis. A NO donor such as sodium nitroprusside (NPS) in the STn before CChrc stimulation increased arterial glucose level and significantly decreased BGR. NPS also induced a higher FOS-ir expression in STn neurons when compared to neurons in control rats that only received artificial cerebrospinal fluid (aCSF) before CChrc stimulation. In contrast, a selective NOS inhibitor such as Nomega-nitro-L-arginine methyl ester (L-NAME) in the STn before CChrc stimulation resulted in an increase of both, systemic glucose and BGR above control values. In this case, the number of FOS-ir positive neurons in the STn decreased when compared to control or to NPS experiments. FOS-ir expression in brainstem cells suggests that CChrc stimulation activates nitroxidergic pathways in the STn to regulate peripheral and central glucose homeostasis. The study of these functionally defined cells will be important to understand brain glucose homeostasis.

Biweekly paclitaxel plus gemcitabine in advanced breast cancer: phase II trial and predictive value of HER2 extracellular domain.

BACKGROUND: We wanted to assess the toxicity and efficacy of paclitaxel plus gemcitabine in advanced breast cancer and to confirm whether circulating HER2 extracellular domain (ECD) correlates with treatment response. PATIENTS AND METHODS: Forty-three patients received paclitaxel 150 mg/m2 followed by gemcitabine 2500 mg/m2, both on day 1 of 14-day cycles, with a maximum of eight cycles. Serum levels of HER2 ECD were assessed by ELISA. RESULTS: All patients were evaluable for toxicity and 42 for efficacy. Overall toxicity was low. Grade 3 neutropenia occurred in 12% of patients and grade 4 in 17%, and other grade 3 toxicities in <5%. One patient had an allergic infusion reaction. Overall response rate was 71% [95% confidence interval (CI) 62% to 81%], with 11 patients achieving a complete response (26%). With a median follow-up of 26 months, the median time to progression was 16.6 months. Response rate correlated significantly with HER2 ECD, with 42% of HER2 ECD-positive patients responding versus 83% of HER2 ECD-negative patients (P = 0.02). Furthermore, response duration was shorter in patients with positive HER2 ECD levels (7.9 versus 14.4 months; P = 0.04). CONCLUSIONS: Paclitaxel plus gemcitabine given as an every 2-weeks schedule is a well tolerated and active regimen in advanced breast carcinoma. This is an attractive combination to use when anthracyclines are not indicated, such as in HER2 positive cases that receive trastuzumab. In addition, elevated levels of HER2 ECD adversely affect the efficacy of treatment.

Prognostic value of cytosolic p53 protein in breast cancer.

The prognostic value of cytosolic p53 protein was evaluated in 458 operable breast carcinomas by immunoblotting using the monoclonal antibody PAb 1801. Two hundred and five carcinomas (45%) showed positive p53 accumulation and 55% were p53 negative. When comparing p53 positivity and other clinicopathological parameters, significant differences were found with younger age (p = 0.017), premenopausal status (p = 0.003), increasing tumor size (p = 0.026), negative estrogen receptor (p = 0.003) and negative progesterone receptor (p = 0.047), but not with histologic grade, axillary lymph node status, stage or erbB-2 expression. With a median follow-up of 34 months (range 3-70), relapse has occurred in 73 patients. Disease-free survival curves showed that patients with p53-positive tumors had a significantly shorter disease-free survival than patients with p53-negative carcinomas (log-rank test, p = 0.027). A multivariate analysis of disease-free survival showed that p53, tumor size, histologic grade and progesterone receptor had significant independent prognostic value. The immunoblotting technique was controlled with p53 immunohistochemistry in 94 paired samples. We obtained a statistically significant correlation (p = 0.0004) between the two methods. Our results show that the immunoblotting technique offers an alternative approach in evaluating the p53 status of breast biopsy material using cytosolic extracts, and confirm that p53 accumulation is a significant independent indicator of a poor prognosis in operable breast carcinoma.

Effects of gamma-linolenic acid and oleic acid on paclitaxel cytotoxicity in human breast cancer cells.

It has been suggested that dietary interventions may improve the effectiveness of cancer chemotherapy. We have examined the combined in vitro cytotoxicity of paclitaxel and the fatty acids gamma-linolenic acid (GLA, 18:3n-6) and oleic acid (OA, 18:1n-9) in human breast carcinoma MDA-MB-231 cells. The effect of fatty acids on paclitaxel chemosensitivity was determined by comparing IC(50) and IC(70) (50 and 70% inhibitory concentrations, respectively) obtained when the cells were exposed to IC(50) and IC(70) levels of paclitaxel alone and fatty acids were supplemented either before or during the exposure to paclitaxel. The 3-4,5-dimethylthiazol-2-yl-2,5-diphenyl-tetrazolium bromide (MTT) assay was used to determine cell growth inhibition. GLA by itself showed antiproliferative effects, and a possible GLA-paclitaxel interaction at the cellular level was assessed by the isobologram and the combination-index (CI) methods. Isobole analysis at the isoeffect levels of 50 and 70% revealed that drug interaction was predominantly synergistic when GLA and paclitaxel were added concurrently for 24 h to the cell cultures. Interaction assessment using the median-effect principle and the combination-index (CI) method showed that exposure of MDA-MB-231 cells to an equimolar combination of concurrent GLA plus paclitaxel for 24 h resulted in a moderate synergism at all effect levels, consistent with the results of the isobologram analysis. When exposure to GLA (24 h) was followed sequentially by paclitaxel (24 h) only an additive effect was observed. The GLA-mediated increase in paclitaxel chemosensitivity was only partially abolished by Vitamin E, a lipid peroxidation inhibitor, suggesting a limited influence of the oxidative status of GLA in achieving potentiation of paclitaxel toxicity. When OA (a non-peroxidisable fatty acid) was combined with paclitaxel, an enhancement of chemosensitivity was found when OA was used concurrently with paclitaxel, although less markedly than with GLA. Pretreatment of MDA-MB-231 cells with OA for 24 h prior to a 24 h paclitaxel exposure produced greater enhancement of paclitaxel sensitivity at high OA concentrations than the concurrent exposure to OA and paclitaxel. The OA-induced sensitisation to paclitaxel was not due to the cytoxicity of the fatty acid itself. When these observations were extended to three additional breast carcinoma cell lines (SK-Br3, T47D and MCF-7), simultaneous exposure to GLA and paclitaxel also resulted in synergism. GLA preincubation followed by paclitaxel resulted in additivity for all cell lines. Simultaneous exposure to paclitaxel and OA enhanced paclitaxel cytotoxicity in T47D and MCF-7 cells, but not in SK-Br3 cells, whereas preincubation with OA failed to increase paclitaxel effectiveness in all three cell lines. For comparison, the effects of other fatty acids on paclitaxel chemosensitivity were examined: GLA was the most potent at enhancing paclitaxel cytotoxicity, followed by alpha-linolenic acid (ALA; 18:3n.3), eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), whereas linoleic acid (LA; 18:2n-6) did not increase paclitaxel toxicity. These findings provide experimental support for the use of fatty acids as modulators of tumour cell chemosensitivity in paclitaxel-based therapy.

Circulating HER2 extracellular domain and resistance to chemotherapy in advanced breast cancer.

To test the hypothesis of an association between HER2 and chemotherapy resistance, we performed a prospective assessment of the predictive value of the circulating HER2 extracellular domain (ECD) in patients with advanced breast carcinoma in the setting of a multicenter Phase II trial using paclitaxel and doxorubicin. Serum samples were collected from 58 patients with metastatic breast carcinoma before first-line chemotherapy for advanced disease, and the levels of circulating HER2 ECD were measured using an enzyme immunoassay. Immunohistochemistry with anti-HER2 monoclonal antibody CB11 was used to assess the overexpression of HER2 in the primary tumors. When 450 fmol/ml was used as a cutoff, 24 cases (41%) had elevated HER2 ECD levels. Elevated levels of circulating HER2 ECD were associated with the expression of HER2 in the primary tumor tissue and with the metastatic tumor burden (evaluated with the marker CA 15-3; P = 0.032 and P = 0.002, respectively) but not with variables such as menopausal status, stage at diagnosis, previous adjuvant therapy, or the number of metastatic sites. The levels of circulating HER2 ECD correlated inversely with the response to treatment. The probability of obtaining a complete response to chemotherapy was significantly lower (P = 0.021) in patients with elevated HER2 ECD levels (0%; 95% confidence interval, 0-13%) compared with patients with nonelevated HER2 (26%; 95% confidence interval, 12-45%). In addition, the duration of clinical response was significantly shorter in patients with elevated HER2 ECD, compared with the cases with nonelevated HER2 (7.5 versus 11 months; P = 0.035). In conclusion, elevated levels of circulating HER2 ECD in patients with metastatic breast cancer correlate with reduced efficacy of a paclitaxel-doxorubicin chemotherapy combination. We suggest that the poor response rate associated with HER2 expression in advanced breast cancer may not be reversed by aggressive chemotherapy alone.

The submental route revisited using the laryngeal mask airway: a technical note.

The submental route for endotracheal intubation is well known and this paper reports the use of the same route for the laryngeal mask airway. This technique can be used whenever it is considered too awkward to perform submental, transoral or transnasal endotracheal intubations. When the surgery has been completed, the mouth is left open to allow the laryngeal mask to be removed; it cannot be left in place in cases of intermaxillary fixation. It is necessary to detach the laryngeal mask orally, never submentally, as it is impossible to remove the mask via the submental route.

Colangitis aguda supurada: experiencia con tratamiento endoscópico / Acute purulent cholangitis: experience with endoscopic treatment

La colangitis aguda es una enfermedad del tracto biliar de alta mortalidad. Su tratamiento debe ser urgente en orden de impedir la sepsis como una de sus complicaciones. La coledocolitiasis es la causa más común de la obstrucción biliar y de la colangitis. El tratamiento quirúrgico y el drenaje percutáneo transhepático están asociados con un alto riesgo de morbilidad y mortalidad. El manejo endoscópico aparece como el mejor procedimiento para solucionar esta condición. Entre Marzo de 1997 y Agosto de 1998, se realizaron 1.314 procedimientos de cirugía endoscópica en vía biliar. 71 (5,4 por ciento) de los pacientes tenían una colangitis aguda, con material purulento o cultivo positivo obtenido durante la terapia descompresiva. 61 por ciento de sexo femenino y una edad promedio de 68 años (rango de 33 a 95). Un 65 por ciento fueron pacientes mayores de 60 años. La obstrucción biliar era debida a coledocolitiasis en el 43,6 por ciento de los pacientes, 40,8 por ciento debida a neoplasia y 5,6 por ciento por estenosis benigna. La colangitis estuvo presente en el 7,5 por ciento de las coledocolitiasis, en 12,5 por ciento de las estenosis malignas y 3,7 por ciento de las benignas. El ultrasonido confirmó sólo el 58 por ciento de las dilataciones de la vía biliar, 12,9 por ciento de las litiasis de la vía biliar y 24,1 por ciento de las dilataciones neoplásicas. Se logró el drenaje de la vía biliar en el 98,5 por ciento de los casos. Sólo en un paciente no se logró solucionar la obstrucción. Fue sometido a cirugía. La esfinterotomía fue realizada en 74,1 por ciento de las obstrucciones litiásicas y en el 61,2 por ciento los cálculos fueron extraídos completamente, mientras el 12,9 por ciento fueron removidos sólo parcialmente. En 25,8 por ciento de pacientes muy críticos fueron tratados en primera instancia con endoprótesis o drenaje nasobiliar. En el 41 por ciento de los casos el cálculo era mayor de 15 mm. Las obstrucciones malignas fueron tratadas con endoprótesis múltiples dejadas por largo plazo. La morbilidad en nuestra serie fue de 2,8 por ciento y la mortalidad de 1,4 por ciento, con un promedio de estadía hospitalaria de 8,8 días. El tratamiento endoscópico de la colangitis por lo tanto, parece como eficiente, seguro y en nuestra opinión, es el mejor método para solucionar esta enfermedad

Angiogenin expression and prognosis in primary breast carcinoma.

Angiogenin is a protein originally isolated as an inducer of new blood vessel growth, and it has been reported to be an effective substrate for tumor cell adhesion. To understand the role of angiogenin in cancer progression, we evaluated the expression of angiogenin in 459 cases with primary breast carcinoma and in 40 benign breast specimens using an immunoassay. Higher angiogenin concentrations were observed in carcinomas in comparison with fibrocystic disease (mean, 17.3 versus 10.9 ng/mg; P = 0.008), but not with fibroadenomas. We selected 5 ng/mg cytosol protein of angiogenin as the normal cutoff for primary breast carcinoma. Eighty-eight percent of carcinomas expressed elevated angiogenin levels and 12% had low levels. We observed an association between elevated levels of angiogenin and low/ moderate histological grade (P = 0.001) and small tumor size (P = 0.026), but not with age, menopausal status, lymph node status, stage of disease, or hormonal receptor status. With a median follow-up of 31 months, breast cancer patients with elevated angiogenin levels had significantly longer disease-free survival (DFS) than patients with low angiogenin (log-rank, P = 0.003). This effect was equally observed in node-negative and node-positive cases. In a multivariate analysis of DFS, only angiogenin, tumor size, and histological grade showed statistical significance. A multivariate analysis of overall survival showed that angiogenin and tumor size were the only significant variables. Serum samples from the breast cancer patients at the time of surgery were available in 194 cases. We evaluated the levels of circulating angiogenin using the same immunoassay as in tumor tissue. Serum angiogenin levels were higher in cancer patients than in 40 healthy controls (mean, 401.2 versus 206.0 ng/ml; P < 0.0001). In breast cancer patients, we observed no correlation between the serum concentrations and the tissue levels of angiogenin (r = 0.115; P = 0.110). In addition, serum levels of angiogenin did not have a prognostic impact on the DFS of breast cancer patients (log-rank, P = 0.581). Our results indicate that elevated levels of tissue angiogenin, but not of circulating angiogenin, are a favorable prognostic factor in primary breast carcinoma, which is consistent with a role of angiogenin as a cancer cell substrate.

Cytogenetic studies in seventy-six cases of B-chronic lymphoproliferative disorders.

The results of cytogenetic studies are reported in 76 patients with B-chronic lymphoproliferative disorders (B-CLPD): 60 patients with chronic lymphocytic leukemia (CLL), six with follicular lymphoma in leukemic phase (FLLP), five with splenic B-cell lymphoma with villous lymphocytes (SLVL), two with chronic prolymphocytic leukemia (CPL), two with hairy cell leukemia (HCL), and one with plasma cell leukemia (PCL). PHA (phytohemagglutinin), PWM (pokeweed mitogen), LPS (lipopolysaccharide from Escherichia Coli), TPA (phorbol 12-myristate acetate), IL6 (interleukin 6), and DxS (dextran sulfate) were used as mitogens. Mitoses were obtained in 75 cases. Clonal aberrations could be demonstrated in 34 cases (44%). In CLL, classical type, chromosomes 6, 11, and 13 were more frequently involved, whereas trisomy 12 was frequently found in CLL mixed-cell type, in FLLP, and CPL. In SLVL the deletion del(7)(q32) is noteworthy and miscellaneous chromosome abnormalities in the remaining patients were observed. Regarding the efficiency of mitogens, PHA turned to be the most effective in obtaining metaphases and in detecting clonal chromosomal aberrations.

Pituitary and adrenals are required for hyperglycemic reflex initiated by stimulation of CBR with cyanide.

We have previously shown that stimulation of carotid body receptors (CBR) with sodium cyanide (NaCN) elicits a rapid hyperglycemic reflex. Here we explore whether the pituitary and adrenals, two glands involved in glucose homeostasis, are necessary for this reflex. Experiments were performed on anesthetized rats that were artificially ventilated. Measurements of hepatic venous-arterial glucose difference indicated that CBR stimulation with a bolus of 5 micrograms/100 g NaCN produced an immediate increase in the output of glucose by the liver. The same dose of NaCN failed to increase hepatic output of glucose in rats with bilateral adrenalectomy or in rats 1 wk after surgical removal of neurohypophysis. Reflex glucose output by the liver was maintained after adenohypophysectomy or in adrenalectomized rats after adrenal autotransplantation to epiploon. Measurements of epinephrine in plasma and in the grafted adrenal tissue showed that the adrenal autograft can store and secrete catecholamines Immunocytochemical observations indicated that the grafted adrenals retain medullary cells. These results indicate that neurohypophysis and adrenals are necessary for the hyperglycemic reflex initiated by CBR stimulation with NaCN and that the participation of these two organs in this reflex is probably humoral.

Low levels of basic fibroblast growth factor (bFGF) are associated with a poor prognosis in human breast carcinoma.

It has been suggested that angiogenesis and angiogenic factors may be strong predictors of relapse in patients with breast carcinoma. We measured the levels of the angiogenic peptide basic fibroblast growth factor (bFGF) in 140 breast tumour cytosols using an immunoassay. There were no significant differences in bFGF levels between breast non-malignant lesions and primary carcinomas. In 124 cases with primary breast cancer, we observed an association of low bFGF levels (< 400 pg mg[-1]) with increasing tumour size (P = 0.023) and stage of disease (P = 0.002). bFGF levels did not correlate with other variables, including axillary nodes, hormone receptors, cathepsin D and the serum tumour markers CA15.3 and CEA. With a median follow-up of 44.0 months, breast cancer patients with low levels of bFGF had a significantly shorter disease-free survival (DFS) than patients with elevated bFGF (log-rank, P < 0.0001). In a multivariate analysis of DFS, only bFGF, T-stage and histological grade showed statistical significance. In a parallel evaluation of circulating bFGF, we did not observe a correlation between the serum and tissue bFGF levels in the 29 selected cases with matched determinations. Our results indicate that low bFGF levels in breast carcinoma are an independent prognostic indicator of poor prognosis and disease recurrence.

Immobilization of Candida rugosa lipase and some properties of the immobilized enzyme.

Lipase (triacylglycerol ester hydrolase, E.C.3.1.1.3) from Candida rugosa has been immobilized on commercially available microporous polypropylene. The enzyme was rapidly adsorbed on the support, and more than 60% of the soluble activity disappeared from the medium after 1 min of incubation at room temperature. A recovery of immobilized activity of 21% was obtained when the wet preparation was immediately assayed with olive oil at the end of the immobilization protocol. The activity of the immobilized enzyme drastically decreased with the loss of water of the preparation. Pretreatment of the support with organic solvents significantly increased the recovered immobilized activity. Our results strongly suggest that the soluble lipase could exist in different aggregation forms depending on the pH of the medium. At acidic pH, the relative proportion of high-molecular-weight forms of the enzyme is higher than at pH 7.0, suggesting that the lipase would be also immobilized in different aggregation forms depending on the pH used in the immobilization procedure. Crosslinking of the adsorbed enzyme with glutaraldehyde diminished its activity but increased the stability of the lipase against the washing-out effect of Triton X-100. Data on the most relevant catalytic properties of the soluble and immobilized enzyme, such as optimum pH and temperature as well as ranges of stability, kinetic parameters, and activation energy for the hydrolysis of olive oil and p-nitrophenyl acetate, are reported.

A new chromosomal anomaly associated with mature B-cell chronic lymphoproliferative disorders: del(7)(q32).

Among 63 patients with chronic lymphoproliferative disorders (CLPD) studied cytogenetically in our laboratory, four showed a del(7)(q32); in two it was the sole cytogenetic anomaly and in two it was part of a complex karyotype. We suggest that despite the rarity of this anomaly, it could be related to CLPD.

New chromosomal abnormality. t(1;19;?) in a case of B-chronic lymphocytic leukemia.

Cytogenetic analysis was performed on peripheral blood cells stimulated with interleukin 6 (IL-6), lipopolysaccharide from Escherichia coli (LPS), phytohemagglutinin (PHA), pokeweed mitogen (PWM) and tetradecanoyl-phorbol-acetate (TPA), in a patient with B-chronic lymphocytic leukemia, showing a t(1;19;?) translocation as the sole abnormality. To our knowledge, this translocation has not been described before in any human neoplasia. In this case, the poor response to therapy (survival time 4 months) suggested that t(1;19;?) could be related to an aggressive course of the disease.