RNA-based determination of ESR1 and HER2 expression and response to neoadjuvant chemotherapy.

BACKGROUND: Hormone and human epidermal growth factor receptor 2 (HER2) receptors are the most important breast cancer biomarkers, and additional objective and quantitative test methods such as messenger RNA (mRNA)-based quantitative analysis are urgently needed. In this study, we investigated the clinical validity of RT-PCR-based evaluation of estrogen receptor (ESR1) and HER2 mRNA expression. PATIENTS AND METHODS: A total of 1050 core biopsies from two retrospective (GeparTrio, GeparQuattro) and one prospective (PREDICT) neoadjuvant studies were evaluated by quantitative RT-PCR for ESR1 and HER2. RESULTS: ESR1 mRNA was significantly predictive for reduced response to neoadjuvant chemotherapy in univariate and multivariate analysis in all three cohorts. The complete pathologically documented response (pathological complete response, pCR) rate for ESR1+/HER2- tumors was 7.3%, 8.0% and 8.6%; for ESR1-/HER2- tumors it was 34.4%, 33.7% and 37.3% in GeparTrio, GeparQuattro and PREDICT, respectively (P < 0.001 in each cohort). In the Kaplan-Meier analysis in GeparTrio patients with ESR1+/HER2- tumors had the best prognosis, compared with ESR1-/HER2- and ESR1-/HER2+ tumors [disease-free survival (DFS): P < 0.0005, overall survival (OS): P < 0.0005]. CONCLUSIONS: Our results suggest that mRNA levels of ESR1 and HER2 predict response to neoadjuvant chemotherapy and are significantly associated with long-term outcome. As an additional option to standard immunohistochemistry and gene-array-based analysis, quantitative RT-PCR analysis might be useful for determination of the receptor status in breast cancer.

[HES 130 as a continuous supplement for organ culture of human corneas?]. / HES 130 als Dauerzusatz in der Organkultur humaner Hornhäute?

BACKGROUND: During organ culture, corneas swell and have to be de-swollen prior to transplantation using a medium. Both swelling and de-swelling lead to increased endothelial cell loss. Furthermore, dextran, the standard de-swelling substance, is toxic and deposited in the cornea. Whether HES is suitable as a continuous supplement for organ culture and keeps the corneas thin was investigated in this study. METHODS: Forty-five human corneas were stored in MEM plus 7.5% HES 130 for 7, 15, 21, and 28 days. Endothelial cell count was determined before and after organ culture. After storage the water content and amount of adenosine phosphates were measured. RESULTS: Water content was 82.29% after 7 days, 82.98% after 15 days, 81.15% after 21 days, and 83.21% after 28 days. The endothelial cell count decreased by 1.20% after 7 days, 0.60% after 15 days, 4.29% after 21 days, and 6.89% after 28 days. ATP was 0.159 micromol/g dry weight after 28 days of organ culture. CONCLUSION: The water content of corneas stored in HES-containing medium remained constant even after 21 days of organ culture. Endothelial cell loss was less than in comparable studies using a standard medium containing 10% FCS; however, ATP concentrations were worse. HES 130 keeps the corneas thin and can be used as a continuous supplement. It facilitates the handling of corneas during organ culture, making dehydration before transplantation unnecessary, and thus also reduces potential stress factors for the cornea.

Two modes of transfer in artificial grammar learning.

Participants can transfer grammatical knowledge acquired implicitly in 1 vocabulary to new sequences instantiated in both the same and a novel vocabulary. Two principal theories have been advanced to account for these effects. One suggests that sequential dependencies form the basis for cross-domain transfer (e.g., Z. Dienes, G. T. M. Altmann, & S. J. Gao, 1999). Another argues that a form of episodic memory known as abstract analogy is sufficient (e.g., L. R. Brooks & J. R. Vokey, 1991). Three experiments reveal the contributions of the 2. In Experiment 1 sequential dependencies form the only basis for transfer. Experiment 2 demonstrates that this process is impaired by a change in the distributional properties of the language. Experiment 3 demonstrates that abstract analogy of repetition structure is relatively immune to such a change. These findings inform theories of artificial grammar learning and the transfer of grammatical knowledge.

Incremental interpretation at verbs: restricting the domain of subsequent reference.

Participants' eye movements were recorded as they inspected a semi-realistic visual scene showing a boy, a cake, and various distractor objects. Whilst viewing this scene, they heard sentences such as 'the boy will move the cake' or 'the boy will eat the cake'. The cake was the only edible object portrayed in the scene. In each of two experiments, the onset of saccadic eye movements to the target object (the cake) was significantly later in the move condition than in the eat condition; saccades to the target were launched after the onset of the spoken word cake in the move condition, but before its onset in the eat condition. The results suggest that information at the verb can be used to restrict the domain within the context to which subsequent reference will be made by the (as yet unencountered) post-verbal grammatical object. The data support a hypothesis in which sentence processing is driven by the predictive relationships between verbs, their syntactic arguments, and the real-world contexts in which they occur.

A new radiographic procedure for obtaining correction factors of 3H-beta-self-absorption for quantitative tritium autoradiography.

A 3H-radiographic method based on the absorption of 3H-beta-particles by an overlying tissue section was established for obtaining correction factors of 3H-beta-absorption (c.f.s) on the cellular level for all kinds of sections in a simple and more precise way as possible with interferometry. Unlabelled paraffin or Araldite sections were mounted on a thin uniformly 3H-labelled section of resin, and autoradiographs were prepared. Grain densities of neuronal cell types and cell-free areas within and outside the paraffin or Araldite sections were evaluated in autoradiographs, where the exposure time or the thickness of the overlying histological section was varied. From these values c.f.s were calculated applying the Beer-Lambert law. It was shown that corresponding c.f.s determined with this new radiographic method correspond well with each other. However, they will only agree with those c.f.s obtained by interferometry, if relative c.f.s are compared. Since the c.f.s are quite sensitive to the section thickness, a new parameter phi was introduced, which helps to assess whether the microtome used works exactly. Generally, the method presented can be used on the cytological level as well as for whole areas in every other autoradiographic study.

Ambiguity in sentence processing.

As listeners and readers, we rarely notice the ambiguities that pervade our everyday language. When we hear the proverb `Time flies like an arrow' we might ponder its meaning, but not the fact that there are almost 100 grammatically permissible interpretations of this short sentence. On occasion, however, we do notice sentential ambiguity: headlines, such as `Two Sisters Reunited After 18 Years in Checkout Counter', are amusing because they so consistently lead to the unintended interpretation (presumably, the sisters did not spend 18 years at the checkout). It is this consistent preference for one interpretation-and one grammatical structure-rather than another that has fuelled research into sentence processing for more than 20 years. Until relatively recently, the dominant belief had been that these preferences arise from general principles that underlie our use of grammar, with certain grammatical constructions being preferred over others. There has now accrued, however, a considerable body of evidence demonstrating that these preferences are not absolute, but can change in particular circumstances. With this evidence have come new theories of sentence processing, some of which, at first glance, radically question the standard notions of linguistic representation, grammar and understanding.

Stages of intestinal carcinogenesis and their control by host NK cells. Implications to prevention and therapy.

DMH-induced (25mg/kg/wk) rodent intestinal carcinogenesis was re-examined using histometry under various conditions including modulation of host natural killer (NK) cell activity and mutagen administration. Early lesion was enterocyte hyperplasia all along the intestinal tract. Cell kinetic analysis showed that it was caused by a slightly altered but still functional "initiated" enterocyte population added to the normal population which remained unchanged. Primarily, initiated stem (IS) cells were added which then produced their initiated progeny which still renewed normally. NK cells countered the initiated cells selectively, either inhibiting IS cell proliferation or killing them when activated by lymphokines. Evidence was obtained that the IS cells could further transform into preneoplastic stem (PS) and then neoplastic stem (NS) cells under the influence of promoters and mutagens, respectively. Subsequent transformation of normal stem cells into IS, PS, and NS cells apparently is the basis of carcinogenesis. These produce lesions, hyperplastic, preneoplastic, and neoplastic, respectively, only when and where NK activity is inhibited. Under the influence of normal NK cells, they remain dormant (nonproliferative), compatible with normal life.

Initiated stem cells in murine intestinal carcinogenesis: prolonged survival, control by NK cells, and progression.

Weekly injections of dimethylhydrazine (DMH) (25 mg/kg), or azoxymethane (AOM) (8 mg/kg) to young adult male CDI mice for 1-2 months produced generalized intestinal crypt hyperplasia, which we measured in duodenum in terms of number of interphase and mitotic cells present in crypts. As shown earlier, the crypts expanded because of the presence of a hyperproliferative "initiated" crypt subpopulation which was also sensitive to natural killer (NK) cells. Hyperplasia was thus present as long as NK activity was suppressed by the carcinogen treatment. After interruption of the treatment for periods of 1, 2, 3, 6 and 10 months in the various groups, hyperplasia soon regressed as a result of elimination of the subpopulation by the recovering NK cells. When NK activity was once again eliminated during the terminal days of these "interruption periods" (by injections of anti-asialo GM-I antibody, alpha AGM-I), the original hyperplasia was fully reconstituted, apparently from stem cells of the subpopulation which survived up to 10 months in their crypt base location. These "initiated stem cells" represented, then, the original carcinogenic insult during the pre-cancerous period. They also appeared to be the source of the eventual neoplasia, as treating the animals with mutagens during the interruption periods produced specific changes in crypt base histology: new "crypt base basophilic" (CBB) cells appeared which produced large accumulations as well as microscopic tumors when NK activity was suppressed (by alpha AGM-I). Some of the initiated stem cells were apparently transformed into neoplastic ones which remained under NK control, the NK cells preventing the establishment of their progeny. Further experiments indicated that, although the initiated stem cells are not eliminated by normal NK activity, activated NK cells can kill them, thereby eliminating the potential source of neoplasia.

Hyperplasia of mouse duodenal crypts and its control by NK cells during the initial phase of DMH carcinogenesis.

The possible regulatory role of NK cells on early events in chemical carcinogenesis remains undefined. The present study examined whether NK cells control 1,2-dimethylhydrazine (DMH)-induced hyperplasia of the duodenal crypt in CD1 mice. Mice receiving chronic DMH treatment showed a dose-dependent hyperplasia confined to the proliferative zone, with a parallel increase in mitotic and 3H-TdR-labelled cells and significant suppression of splenic NK activity. Complete ablation of splenic NK activity with anti-asialo GM-I antibody (alpha AGM-I) treatment slightly enhanced hyperplasia. Halving of the DMH dose for 2 weeks led to regression of hyperplasia, which was totally prevented by alpha AGM-I treatment. The alpha AGM-I treatment alone did not influence crypt size in normal mice. Finally, a stimulation of NK activity with Poly I:C treatment in DMH-treated mice caused regression of the DMH-induced hyperplasia. Our results suggest that hyperplastic cells with possible genetic alterations induced by the carcinogen express target structures for NK cells, but that simultaneous carcinogen-induced suppression of NK activity hampers their containment, allowing progression of hyperplasia to neoplasia, possibly owing to additional genetic changes.

Behavior of fetal intestinal organ culture explanted onto a collagen substratum.

A model of organ culture of 18 day old fetal rat intestine (Quaroni, 1985) was modified and characterized in the present work with the purpose of developing an in vitro model for the study of intestinal epithelial cell behaviour. Fragments of this intestine were kept in suspension culture for 7 days and then explanted onto collagen (type I) matrix. Within a day, the fragments became anchored to the substratum and a circular monolayer grew out to about 1 cm diameter. In the fragments, an outer layer of absorptive epithelial cells came to enclose a stroma, which was polarized into a loose (mesenchymal) and a dense portion. The dense portion contained a mixture of smooth muscle cells and primitive stem-type epithelial cells ('p-cells'). After explantation, at the contact point with the matrix, the epithelium broke up and the mesenchyme grew into the matrix and anchored the fragment. The epithelial edges now became continuous with the developing monolayer. Radioautography with tritiated thymidine indicated a constant cell renewal in epithelium and monolayer apparently from foci of p-cells, a reserve population of which was seen to be sequestered among the smooth muscle cells. Activated stem cells could differentiate into three mature epithelial phenotypes, each differentiation pathway apparently being determined by the type of underlying stroma. Immunohistochemistry using gold- and fluorescein-labeled monoclonal antibodies indicated that adult differentiation-specific markers (e.g. brush border enzymes) were present in the fragment epithelium but not in the monolayer cells. On the other hand, the monolayer cells could be induced to express some of these markers by contact with mesenchymal cells or by co-culturing with fibroblastic cell lines. Matrigel substratum mixed with collagen (type I) supported the appearance in monolayer of strands positive for amino-peptidase and lactase. The model thus appears to be suitable for the in vitro study of epithelial renewal and differentiation, and it has already provided some results in this respect.

[Viewpoints on preparations for integrating data processing systems in routine microbiology diagnosis]. / Gesichtspunkte der Einsatzvorbereitung von Datenverarbeitungssystemen (DV-Systemen) in der mikrobiologischen Routinediagnostik.

The enormously risen and further increasing numbers of examinations and tests in microbiological diagnostics within the last years need new methods for treatment. One possibility to meet the higher requirements for information of the clinic without loss in quality at constant staff is the integration of the microcomputer technique into the laboratory as direct "tool". Demands for a qualitatively high empirical antimicrobial chemotherapy, chemotherapy according to antibiotic susceptibility tests, indicated use of antimicrobial drugs and control measures of infectious processes in general are met only by means of a fast information processing. The microcomputer technique in the laboratory provides also the chance to automate still manually performed tests and comprises according to algorithm the strict observation of the diagnostic process and its control. The application of the microcomputer technique on the one hand means for the technical assistant the omission of much manually performed work, on the other hand enables work of higher quality and supports decisions in the diagnostic process. Mathematical and statistical calculations are no longer connected with great losses of activity. The actual need for information of the clinician is met in time in different ways.

[Prenatal diagnosis and obstetric management in association of hydramnios and homozygosity]. / Pränatale Diagnose und geburtshilfliches Management bei Assoziation von Hydramnion und Homozygotie.

The patient was referred to hospital in the 29th week of gestation owing to polyhydramnions. Ultrasonography revealed isolated left-sided hypertrophy (from foetal chest to the lower extremity) with subcutaneous lymph cysts. Amniocentesis for genetic examination revealed a chromosomal disorder showing homozygous t(13q, 14q) translocation. This extremely rare anomaly could be explained only and was finally proven by a father-daughter incest. The authors underline the necessity of karyotyping as well as the ultrasonic exclusion of foetal malformations in patients with polyhydramnions.

A gradual decrease in nucleolar size with the maturation of columnar epithelial cells in the adult rat intestine under normal and various experimental conditions.

The columnar cells, which form over 90% of the epithelium in the small intestine, undergo rapid and continuous renewal and maturation. Samples from duodenum, jejunum, upper, mid- and terminal ileum of young male rats were processed for histology. The average maximal nucleolar area was determined in 10-cell-wide bands of the basal, mid and upper levels of crypts and villi, respectively, by image analysis; in the duodenum, it was 2.8, 2.1, 1.7, 1.5, 1.3 and 0.8 (in micron2) in the respective epithelial levels from crypt base to villus top. Although villus size decreased by 68% from duodenum to terminal ileum, nucleolar area was similar at each respective epithelial level along the intestine. This indicated that nucleolar size was related to cell maturity, rather than to the size of epithelium. In other groups of rats, the duodenum was examined after administering specific inhibitors. Methotrexate (within a day) and cycloheximide (within 3 h) did not significantly affect nucleolar size, indicating that the decrease in size was not under the influence of immediate synthesis of nucleic acid or protein. On the other hand, tunicamycin (within a day) delayed the decrease and actinomycin D (within 3 h) caused a maximal decrease in all nucleoli. This implied that a glycoprotein factor and some changes in DNA were involved in the decrease in nucleolar size. The rate of protein synthesis in duodenum was then measured by grain count per cell area in autoradiographs made after 1 h of injection of [3H]leucine. From crypt base to villus base, the grain count doubled while the nucleoli decreased to nearly half of their size in the crypt base. When actinomycin D injection preceded the [3H]leucine administration, all nucleoli decreased markedly and the grain counts increased by about 30% in all epithelial levels. It thus appears that the decrease in nucleolar size stimulates protein synthesis, possibly by the release of ribosomal material or some other factor. Protein synthesis in turn has been shown to be related to cell maturation. It is concluded that the nucleolus is involved in some manner in the regulation of the maturation and renewal of the epithelial cells.

Effects of 1,2-dimethylhydrazine on the number of epithelial cells present in the villi, crypts, and mitotic pool along the rat small intestine.

Young adult male rats received 1,2-dimethylhydrazine (25 mg/kg) twice weekly for 2 months and once a week thereafter for up to 6 months. Histological samples of duodenum, jejunum, and upper, mid-, and terminal ileum were prepared from groups killed at each month. Using cell counts, the average number of epithelial cells was determined per representative section of villi and crypts and was used as an index of villus size and crypt size, respectively. The average number of mitotic figures in representative crypt sections was also determined. All three parameters increased during treatment, and the increments showed a specific pattern in relation to time and intestinal region. Mitotic number showed a consistent change all along the small intestine: close to 20% rise by 3 months; decrease to near control level by the fourth month; and a rise thereafter. Probably, a systemic stimulation of mitotic activity by 1,2-dimethylhydrazine took place. The crypt size index changed similarly, showing highly significant correlation with mitotic number. This correlation indicated that average mitotic time and cell cycle time remained unchanged and the number of divisions increased in progenitor cells. Calculations showed that only a fraction of the progenitor cells was involved. These were probably "initiated" cells. There was a net increase of initiated cell numbers with time, but a sharp drop at 4 months indicated that there is a mechanism inducing a regression of the initiated cell population. Villus size increased linearly in the duodenum and jejunum. In the ileum, there was also a net increase but with some initial fluctuation. In general, villus size seemed to increase so as to maintain a fairly stable turnover time. This would mean that the increased mitotic activity was balanced by increasing differentiation.

Citrobacter koseri isolated in Israel, 1972-83.

Cultures of Citrobacter koseri (syn. C. diversus), an opportunistic pathogen that was isolated in Israel during 1972-83, were studied by sero- and biotyping and for susceptibility to antibacterial agents. Of 1,172 cultures, 96% were isolated from adults, more than half from urinary tract infections. Other frequent sources were superficial wounds and discharges from the respiratory and genital tracts. Seventeen strains originated from blood cultures of patients with sepsis and four from the cerebrospinal fluid of newborns with meningitis. The isolates belonged to 17 O groups, 60 serotypes and 5 biotypes. The most frequent serotypes were 15:a:[2], first identified in Israel, and 1:a:[2]. The most frequent biotypes were c and a. No correlation was found between sero- or biotypes and source of isolation or type of disease. Of 720 cultures tested for sensitivity, all were resistant to ampicillin and carbenicillin, but only 9.3% were resistant to one or more of nine other antibacterial agents tested.

Radioautographic visualization of differences in the pattern of [3H]uridine and [3H]orotic acid incorporation into the RNA of migrating columnar cells in the rat small intestine.

The epithelium of rat small intestine was radioautographed to examine whether RNA is synthesized by the salvage pathway as shown after [3H]uridine injection or by the de novo pathway as shown after [3H]orotic acid injection. The two modes of RNA synthesis were thus investigated during the migration of columnar cells from crypt base to villus top, and the rate of synthesis was assessed by counting silver grains over the nucleolus and nucleoplasm at six levels along the duodenal epithelium--that is, in the base, mid, and top regions of the crypts and in the base, mid, and top regions of the villi. Concomitant biochemical analyses established that, after injection of either [5-3H]uridine or [5-3H]orotic acid: (a) buffered glutaraldehyde fixative was as effective as perchloric acid or trichloracetic acid in insolubilizing the nucleic acids of rat small intestine; (b) a major fraction of the nucleic acid label was in RNA, that is, 91% after [3H]uridine and 72% after [3H]orotic acid, with the rest in DNA; and (c) a substantial fraction of the RNA label was in poly A+ RNA (presumed to be messenger RNA). In radioautographs of duodenum prepared after [3H] uridine injection, the count of silver grains was high over nucleolus and nucleoplasm in crypt base cells and gradually decreased at the upper levels up to the villus base. In the rest of the villus, the grain count over the nucleolus was negligible, while over the nucleoplasm it was low but significant. After [3H]-orotic acid injection, the number of silver grains over the nucleolus was negligible at all levels, whereas over the nucleoplasm the number was low in crypt cells, but high in villus cells with a peak in mid villus. The interpretation is that, except for a small amount of label incorporated into DNA from either precursor by crypt cells, the bulk of the label is incorporated into RNA as follows. In the crypts, cells make almost exclusive use of uridine, that is, of the salvage pathway, for the synthesis of ribosomal RNA in the nucleolus and of messenger and transfer RNA in the nucleoplasm. However, when cells pass from crypt to villus, they mainly utilize orotic acid--i.e., the de novo pathway--for the synthesis of messenger and transfer RNA within the nucleoplasm.