Oncogene-induced basement membrane invasiveness in human mammary epithelial cells

Expression of the intermediate filament protein vimentin, and loss of the cellular adhesion protein uvomorulin (E-cadherin) have been associated with increased invasiveness of established human breast cancer cell linesin vitro andin vivo. In the current study, we have further examined these relationships in oncogenically transformed human mammary epithelial cells. A normal human mammary epithelial strain, termed 184, was previously immortalized with benzo[a]pyrene, and two distinct sublines were derived (A1N4 and 184B5). These sublines were infected with retroviral vectors containing a single or two oncogenes of the nuclear, cytoplasmic, and plasma membrane-associated type (v-ras ^H, v-ras ^Ki, v -mos, SV40T and c -myc). All infectants have been previously shown to exhibit some aspects of phenotypic transformation. In the current study, cellular invasiveness was determinedin vitro using Matrigel, a reconstituted basement membrane extract. Lineage-specific differences were observed with respect to low constitutive invasiveness and invasive changes after infection withras, despite similarras-induced transformation of each line. Major effects on cellular invasiveness were observed after infection of the cells with two different oncogenes (v-ras ^H + SV40T and v -ras ^H + v -mos). In contrast, the effects of single oncogenes were only modest or negligible. All oncogenic infectants demonstrated increased attachment to laminin, but altered secretion of the 72 kDa and 92 kDa gelatinases was not associated with any aspect of malignant progression. Each of the two highly invasive double oncogene transformants were vimentinpositive and uvomorulin-negative, a phenotype indicative of the epithelial-mesenchymal transition (EMT) previously associated with invasiveness of established human breast cancer cell lines. Weakly invasive untransformed mammary epithelial cells in this study were positive for both vimentin and uvomorulin, suggesting that uvomorulin may over-ride the otherwise vimentin-associated invasiveness.     

Reversible inactivation of macaque frontal eye field

 The macaque frontal eye field (FEF) is involved in the generation of saccadic eye movements and fixations. To better understand the role of the FEF, we reversibly inactivated a portion of it while a monkey made saccades and fixations in response to visual stimuli. Lidocaine was infused into a FEF and neural inactivation was monitored with a nearby microelectrode. We used two saccadic tasks. In the delay task, a target was presented and then extinguished, but the monkey was not allowed to make a saccade to its location until a cue to move was given. In the step task, the monkey was allowed to look at a target as soon as it appeared. During FEF inactivation, monkeys were severely impaired at making saccades to locations of extinguished contralateral targets in the delay task. They were similarly impaired at making saccades to locations of contralateral targets in the step task if the target was flashed for ≤100 ms, such that it was gone before the saccade was initiated. Deficits included increases in saccadic latency, increases in saccadic error, and increases in the frequency of trials in which a saccade was not made. We varied the initial fixation location and found that the impairment specifically affected contraversive saccades rather than affecting all saccades made into head-centered contralateral space. Monkeys were impaired only slightly at making saccades to contralateral targets in the step task if the target duration was 1000 ms, such that the target was present during the saccade: latency increased, but increases in saccadic error were mild and increases in the frequency of trials in which a saccade was not made were insignificant. During FEF inactivation there usually was a direct correlation between the latency and the error of saccades made in response to contralateral targets. In the delay task, FEF inactivation increased the frequency of making premature saccades to ipsilateral targets. FEF inactivation had inconsistent and mild effects on saccadic peak velocity. FEF inactivation caused impairments in the ability to fixate lights steadily in contralateral space. FEF inactivation always caused an ipsiversive deviation of the eyes in darkness. In summary, our results suggest that the FEF plays major roles in (1) generating contraversive saccades to locations of extinguished or flashed targets, (2) maintaining contralateral fixations, and (3) suppressing inappropriate ipsiversive saccades. Received: 2 February 1996 / Accepted: 26 February 1997     

Systemic and mucosal antibody responses specific to SARS-CoV-2 during mild versus severe COVID-19

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Bulk and Solution Copolymerization of Butyl Acrylate/Methyl Methacrylate at Elevated Temperatures

A series of bulk and solution (in toluene) copolymerizations of butyl acrylate/methyl methacrylate were performed independently at two laboratories. The runs were at elevated temperatures ranging from 90 to 140 °C conducted to high conversion levels, and samples were characterized for conversion, cumulative copolymer composition and number‐ and weight‐average molecular weights and distribution. Variation of the comonomer feed composition, temperature, and the solvent, initiator and chain transfer agent concentrations was studied. Using a mechanistic model, conversion data were predicted to high conversions using terminal model kinetics at 90 and 115 °C. The copolymer composition data conformed to the terminal kinetic model over the entire temperature range. Solvent effects were reflected by changes in the butyl acrylate rate constants.

Composition vs. conversion. Effect of feed composition for runs at 140 °C.     

Clericuzio type poikiloderma with neutropenia is distinct from Rothmund-Thomson syndrome

Two siblings from a consanguineous family presented with a poikiloderma of limbs and face, plantar keratoderma, and toenail pachyonychia. Neutropenia and neutrophil dysfunction with impairment of the respiratory burst and bacterial killing resulted in frequent respiratory tract infections. A bronchocentric granulomatous pneumonia was a fatal complication. The clinical presentation is consistent with Clericuzio type poikiloderma with neutropenia. Literature review identified several additional probable patients. Genetic linkage analysis excluded the locus of the RECQL4 gene, mutations in which have been described in some patients with the Rothmund-Thomson poikiloderma syndrome. This report confirms the clinical and genetic identity of the Clericuzio type of poikiloderma with neutropenia syndrome.     

Malignant Melanoma of Soft Parts Involving the Head and Neck Region: Review of Literature and Case Report

Malignant melanoma of soft parts (MMSP) was originally described as a distinct entity by Enzinger in 1965 and was termed “clear cell sarcoma of tendons and aponeuroses” because of its association with tenosynovial structures. It has been shown immunophenotypically and ultrastructurally that this tumor is derived from neuroectoderm and shares a number of features with cutaneous melanoma. Over 95% of MMSPs present in the extremities, with the head and neck region (1.9%) being an unusual site. This study presents an additional case of MMSP of the head and neck region involving the posterior cervical region in a 15-year-old Hispanic male and reviews the literature on MMSP. Ultrastructural examination showed rudimentary cell attachments, smooth cell membranes, discontinuous basal lamina, scanty glycogen, and occasional premelanosomes in some tumor cells. Cytogenetic analysis showed a reciprocal translocation between the long arms of chromosomes 12 and 22 [t(12:22)(q13;q12.2)], characteristic for MMSP and not seen in cutaneous melanoma. Survival in MMSP has been correlated with tumor size, tumor necrosis, and ploidy status. Overall reported clinical outcome for this tumor is as follows: died of disease, 45%; alive with disease, 23%; no evidence of disease, 30%; and died of other causes, 2%. MMSP represents a distinct entity with a characteristic ultrastructural appearance and a tumor defining cytogenetic translocation.     

Effects of exercise cessation on lipids and lipoproteins in distance runners and power athletes

Summary The purpose of this study was to determine the effects of short-term exercise cessation on lipid and lipoprotein profile and insulin sensitivity in highly trained runners (n=12; mean age 19.9 years) and power athletes (n=12; mean age 24.4 years). Following 14 days of exercise cessation, running time to exhaustion and maximal oxygen uptake decreased by 9.2% and 4.8% (P < 0.05) in the runners, while in the power athletes one repetition maximum squat and bench press did not change (P>0.05). No changes occurred in body composition. Data from a 2-h oral glucose tolerance test revealed an impairment of the glycemic state in all athletes (P<0.05). In contrast, exercise cessation did not significantly (P>0.05) alter plasma levels of cholesterol, triglycerides, and low density (LDL) and high density lipoprotein (HDL). No changes were observed in HDL₂, HDL_2b, and HDL₃ subfractions, LDL diameter, and qualitative LDL pattern (P>0.05). These data thus suggest that despite a decrease in insulin sensitivity, short-term exercise cessation, independent of exercise mode, was insufficient to alter plasma lipid and lipoprotein profiles in well-trained athletes.     

Molecular epidemiology of Pseudomonas aeruginosa colonization in a burn unit: persistence of a multidrug-resistant clone and a silver sulfadiazine-resistant clone

To study the epidemiology of Pseudomonas aeruginosa colonization in a 32-bed burn wound center (BWC), 321 clinical and 45 environmental P. aeruginosa isolates were collected by prospective surveillance culture over a 1-year period and analyzed by serotyping, drug susceptibility testing, and amplified fragment length polymorphism (AFLP) analysis. Among 441 patients treated at the center, 70 (16%) were colonized with P. aeruginosa, including 12 (17%) patients who were colonized on admission and 58 (83%) patients who acquired the organism during their stay. Of the 48 distinct AFLP genotypes found, 21 were found exclusively in the environment, 15 were isolated from individual patients only, and 12 were responsible for the colonization of 57 patients, of which 2 were also isolated from the environment, but secondary to patient carriage. Polyclonal P. aeruginosa colonization with strains of two to four genotypes, often with different antibiotic susceptibility patterns, was observed in 19 patients (27%). Two predominant genotypes were responsible for recurrent outbreaks and the colonization of 42 patients (60% of all colonized patients). The strain with one of those genotypes appeared to be endemic to the BWC and developed multidrug resistance (MDR) at the end of the study period, whereas the strain with the other genotype was antibiotic susceptible but resistant to silver sulfadiazine (SSD(r)). The MDR strain was found at a higher frequency in sputum samples than the SSD(r) strain, which showed a higher prevalence in burn wound samples, suggesting that anatomic habitat selection was associated with adaptive resistance to antimicrobial drugs. Repeated and thorough surveys of the hospital environment failed to detect a primary reservoir for any of those genotypes. Cross-acquisition, resulting from insufficient compliance with infection control measures, was the major route of colonization in our BWC. In addition to the AFLP pattern and serotype, analysis of the nucleotide sequences of three (lipo)protein genes (oprI, oprL, and oprD) and the pyoverdine type revealed that all predominant strains except the SSD(r) strain belonged to recently identified clonal complexes. These successful clones are widespread in nature and therefore predominate in the patient population, in whom variants accumulate drug resistance mechanisms that allow their transmission and persistence in the BWC.     

Cytokine gene expression in peripheral blood mononuclear cells and tissues of cattle infected with Mycobacterium avium subsp. paratuberculosis: evidence for an inherent proinflammatory gene expression pattern

In cattle and other ruminants, infection with the intracellular pathogen Mycobacterium avium subsp. paratuberculosis results in a granulomatous enteritis (Johne's disease) that is often fatal. The key features of host immunity to M. avium subsp. paratuberculosis infection include an appropriate early proinflammatory and cytotoxic response (Th1-like) that eventually gives way to a predominant antibody-based response (Th2-like). Clinical disease symptoms often appear subsequent to waning of the Th1-like immune response. Understanding why this shift in the immune response occurs and the underlying molecular mechanisms involved is critical to future control measures and diagnosis. Previous studies have suggested that M. avium subsp. paratuberculosis may suppress gene expression in peripheral blood mononuclear cells (PBMCs) from infected cows, despite a continued inflammatory reaction at sites of infection. In the present study, we tested the hypothesis that exposure to M. avium subsp. paratuberculosis suppresses a proinflammatory gene expression pattern in PBMCs from infected cows. To do this, we examined expression of genes encoding interleukin-1alpha (IL-1alpha), IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p35, IL-16, and IL-18, as well as genes encoding gamma interferon (IFN-gamma), transforming growth factor beta (TGF-beta), and tumor necrosis factor alpha (TNF-alpha), in PBMCs, intestinal lesions, and mesenteric lymph nodes of cattle naturally infected with M. avium subsp. paratuberculosis. Cytokine gene expression in these cells and tissues was compared to expression in similar cells and tissues from control uninfected cattle. Our comprehensive results demonstrate that for most cytokine genes, including the genes encoding IFN-gamma, TGF-beta, TNF-alpha, IL-1alpha, IL-4, IL-6, IL-8, and IL-12p35, differential expression in PBMCs from infected and control cattle did not require stimulation with M. avium subsp. paratuberculosis. In fact, stimulation with M. avium subsp. paratuberculosis tended to reduce the differential expression observed in infected and uninfected cows for genes encoding IFN-gamma, IL-1alpha, and IL-6. Only IL-10 gene expression was consistently enhanced by M. avium subsp. paratuberculosis stimulation of PBMCs from subclinically infected cattle. In ileal tissues from M. avium subsp. paratuberculosis-infected cattle, expression of the genes encoding IFN-gamma, TGF-beta, IL-5, and IL-8 was greater than the expression in comparable tissues from control uninfected cattle, while expression of the gene encoding IL-16 was lower in tissues from infected cattle than in control tissues. Mesenteric lymph nodes draining sites of M. avium subsp. paratuberculosis infection expressed higher levels of IL-1alpha, IL-8, IL-2, and IL-10 mRNA than similar tissues from control uninfected cattle expressed. In contrast, the genes encoding TGF-beta and IL-16 were expressed at lower levels in lymph nodes from infected cattle than in tissues from uninfected cattle. Taken together, our results suggest that cells or other mechanisms capable of limiting proinflammatory responses to M. avium subsp. paratuberculosis develop in infected cattle and that a likely place for development and expansion of these cell populations is the mesenteric lymph nodes draining sites of infection.