Effects of double ligation of Stensen's duct on the rabbit parotid gland

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14–30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.     

AN INDIRECT ENZYME-LINKED IMMUNOSORBENT ASSAY FOR THE DETECTION OF LEPTOSPIRA INTERROGANS SEROVAR POMONA ANTIBODIES IN BOVINE SERA

An indirect ELISA was developed and initially evaluated for the detection of bovine antibodies to Leptospira interrogans serovar pomona. The antigen used in this ELISA was extracted from a serovar pomona culture supernatant by a combination of centrifugation, digestion with proteinase K and ultra-centrifugation. The antigen showed little cross-reaction with immune rabbit sera to L. interrogans serovars copenhageni, grippotyphosa, hardjo and sejroe and, Leptospira biflexa serovar patoc. Some cross-reaction was observed with immune rabbit serum to L. interrogans serovar canicola. The relative sensitivity of the ELISA was 94.76% confidence interval =± 3.32%) when estimated with bovine sera (n=172) with serovar pomona microscopic agglutination test (MAT) titers of 100. The relative specificity of the ELISA was 99.28% (95% confidence interval = 1.40%) when estimated with bovine sera (n=139) with MAT titers of <100 to L. interrogans serovars canicola, copenhageni, grippotyphosa, hardjo, pomona and sejroe. Thirty six of 258 field sera (13.95%) with serovar pomona MAT titers of <100, gave positive reactions in the ELISA.     

DEVELOPMENT AND EVALUATION OF A RAPID ENZYME-LINKED IMMUNOFILTRATION ASSAY (ELIFA) AND AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR THE DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN B

An enzyme-linked immunofiltration assay (ELIFA) and a microtitre plate enzyme-linked immunosorbent assay (ELISA) were developed and compared for their ability to detect staphylococcal enterotoxin B (SEB). The double antibody capture format was used for both assays. Factors which improved the sensitivity of the ELIFA system were (1) addition of casein and thimerosal to the antigen dilution buffer; (2) addition of polyethylene glycol (MW 6000) to the detection and conjugate antibody dilution buffers; and (3) washing with diethanolamine buffer prior to addition of the substrate/chromogen. The ELIFA system had a turnaround time of approximately 1 h and a detection limit of 1 ng/mL of purified SEB. The ELISA had a total turnaround time of 21 h, or 3 h using plates pre-coated overnight with the capture antibody. The detection limit of the ELISA for purified SEB was 0.05 ng/mL. The detection limit of SEB in cheese samples spiked with purified enterotoxin and subjected to a simple extraction procedure was 1 ng/mL and 0.1 ng/mL of extract, with the ELIFA and the ELISA, respectively.     

Increased MMPs expression and decreased contraction in the rat myometrium during pregnancy and in response to prolonged stretch and sex hormones

Normal pregnancy is associated with uterine relaxation to accommodate the stretch imposed by the growing fetus; however, the mechanisms underlying the relationship between pregnancy-associated uterine stretch and uterine relaxation are unclear. We hypothesized that increased uterine stretch during pregnancy is associated with upregulation of matrix metalloproteinases (MMPs), which in turn cause inhibition of myometrium contraction and promote uterine relaxation. Uteri from virgin, midpregnant (day 12), and late-pregnant rats (day 19) were isolated, and myometrium strips were prepared for measurement of isometric contraction and MMP expression and activity using RT-PCR, Western blot analysis, and gelatin zymography. Oxytocin caused concentration-dependent contraction of myometrium strips that was reduced in mid- and late-pregnant rats compared with virgin rats. Pretreatment with the MMP inhibitors SB-3CT (MMP-2/MMP-9 Inhibitor IV), BB-94 (batimastat), or Ro-28-2653 (cipemastat) enhanced contraction in myometrium of pregnant rats. RT-PCR, Western blot analysis, and gelatin zymography demonstrated increased mRNA expression, protein amount, and activity of MMP-2 and MMP-9 in myometrium of late-pregnant>midpregnant>virgin rats. Prolonged stretch of myometrium strips of virgin rats under 8 g basal tension for 18 h was associated with reduced contraction and enhanced expression and activity of MMP-2 and MMP-9, which were reversed by MMP inhibitors. Concomitant treatment of stretched myometrium of virgin rats with 17β-estradiol (E2), progesterone (P4), or E2+P4 was associated with further reduction in contraction and increased MMP expression and activity. MMP-2 and MMP-9 caused significant reduction of oxytocin-induced contraction of myometrium of virgin rat. Thus, normal pregnancy is associated with reduced myometrium contraction and increased MMPs expression and activity. The results are consistent with the possibility that myometrium stretch and concomitant increase in sex hormones during pregnancy are associated with increased expression/activity of specific MMPs, which in turn inhibit uterine contraction and promote uterine relaxation.     

A microbiological assay for sterigmatocystin,T-2 toxin and zearalenone

A survey was done to find microorganisms useful for assaying sterigmatocystin; T-2 toxin and zearalenone.Staphylococcus aureus was found to be sensitive to T-2 toxin and zearalenone;Bacillus cereus was found to be sensitive to T-2 toxin only; andEscherichia coli was sensitive to sterigmatocystin. The response of the organisms to sterigmatocystin; T-2 toxin and zearalenone was found to be linear between 4 and 100 μg with sterigmatocystin toE. coli; between 2 and 25 μg with T-2 toxin toStaph, aureus andB. cereus; and between 4 and 100 μg with zearalenone toStaph, aureus. The lower limits of sensitivity of the test were 2 μg T-2 toxin and zearalenone, and 4 μg sterigmatocystin. The assay is rapid (15–17 hrs); simple and inexpensive; and can be used to verify the toxicity of samples and to confirm thin layer chromatographic results.     

Understanding and circumventing resistance to anticancer monoclonal antibodies

With the widespread use of therapeutic monoclonal antibodies in the treatment of patients with cancer, resistance to these agents has become a major issue. Preclinical models of drug action or resistance have contributed to unravel the main mechanisms of resistance, involving both tumor-associated and host related factors. However our understanding of how a monoclonal antibody destroys cancer cells in a patient and why it one day stops being effective are still far from being complete. This review focuses on the available data on mechanisms of action and resistance to rituximab and includes some additional information for other monoclonal antibodies. Innovative approaches designed to overcome resistance, such as combination immunotherapy, costimulation with cytokines or growth factors are presented.Key words: monoclonal antibodies, resistance, rituximab, cetuximab, trastuzumab     

Improved pulmonary vascular reactivity and decreased hypertrophic remodeling during nonhypercapnic acidosis in experimental pulmonary hypertension

Pulmonary hypertension (PH) is characterized by pulmonary arteriolar remodeling with excessive pulmonary vascular smooth muscle cell (VSMC) proliferation. This results in decreased responsiveness of pulmonary circulation to vasodilator therapies. We have shown that extracellular acidosis inhibits VSMC proliferation and migration in vitro. Here we tested whether induction of nonhypercapnic acidosis in vivo ameliorates PH and the underlying pulmonary vascular remodeling and dysfunction. Adult male Sprague-Dawley rats were exposed to hypoxia (8.5% O(2)) for 2 wk, or injected subcutaneously with monocrotaline (MCT, 60 mg/kg) to develop PH. Acidosis was induced with NH(4)Cl (1.5%) in the drinking water 5 days prior to and during the 2 wk of hypoxic exposure (prevention protocol), or after MCT injection from day 21 to 28 (reversal protocol). Right ventricular systolic pressure (RVSP) and Fulton's index were measured, and pulmonary arteriolar remodeling was analyzed. Pulmonary and mesenteric artery contraction to phenylephrine (Phe) and high KCl, and relaxation to acetylcholine (ACh) and sodium nitroprusside (SNP) were examined ex vivo. Hypoxic and MCT-treated rats demonstrated increased RVSP, Fulton's index, and pulmonary arteriolar thickening. In pulmonary arteries of hypoxic and MCT rats there was reduced contraction to Phe and KCl and reduced vasodilation to ACh and SNP. Acidosis prevented hypoxia-induced PH, reversed MCT-induced PH, and resulted in reduction in all indexes of PH including RVSP, Fulton's index, and pulmonary arteriolar remodeling. Pulmonary artery contraction to Phe and KCl was preserved or improved, and relaxation to ACh and SNP was enhanced in NH(4)Cl-treated PH animals. Acidosis alone did not affect the hemodynamics or pulmonary vascular function. Phe and KCl contraction and ACh and SNP relaxation were not different in mesenteric arteries of all groups. Thus nonhypercapnic acidosis ameliorates experimental PH, attenuates pulmonary arteriolar thickening, and enhances pulmonary vascular responsiveness to vasoconstrictor and vasodilator stimuli. Together with our finding that acidosis decreases VSMC proliferation, the results are consistent with the possibility that nonhypercapnic acidosis promotes differentiation of pulmonary VSMCs to a more contractile phenotype, which may enhance the effectiveness of vasodilator therapies in PH.     

A full Bayesian hierarchical mixture model for the variance of gene differential expression

Background  

In many laboratory-based high throughput microarray experiments, there are very few replicates of gene expression levels. Thus, estimates of gene variances are inaccurate. Visual inspection of graphical summaries of these data usually reveals that heteroscedasticity is present, and the standard approach to address this is to take a log₂ transformation. In such circumstances, it is then common to assume that gene variability is constant when an analysis of these data is undertaken. However, this is perhaps too stringent an assumption. More careful inspection reveals that the simple log₂ transformation does not remove the problem of heteroscedasticity. An alternative strategy is to assume independent gene-specific variances; although again this is problematic as variance estimates based on few replications are highly unstable. More meaningful and reliable comparisons of gene expression might be achieved, for different conditions or different tissue samples, where the test statistics are based on accurate estimates of gene variability; a crucial step in the identification of differentially expressed genes.