14-3-3 protein homologs required for the DNA damage checkpoint in fission yeast.

During the cell cycle, DNA is replicated and segregated equally into two daughter cells. The DNA damage checkpoint ensures that DNA damage is repaired before mitosis is attempted. Genetic studies of the fission yeast Schizosaccharomyces pombe have identified two genes, rad24 and rad25, that are required for this checkpoint. These genes encode 14-3-3 protein homologs that together provide a function that is essential for cell proliferation. In addition, S. pombe rad24 null mutants, and to a lesser extent rad25 null mutants, enter mitosis prematurely, which indicates that 14-3-3 proteins have a role in determining the timing of mitosis.

Atm-dependent interactions of a mammalian chk1 homolog with meiotic chromosomes.

BACKGROUND: Checkpoint pathways prevent cell-cycle progression in the event of DNA lesions. Checkpoints are well defined in mitosis, where lesions can be the result of extrinsic damage, and they are critical in meiosis, where DNA breaks are a programmed step in meiotic recombination. In mitotic yeast cells, the Chk1 protein couples DNA repair to the cell-cycle machinery. The Atm and Atr proteins are mitotic cell-cycle proteins that also associate with chromatin during meiotic prophase I. The genetic and regulatory interaction between Atm and mammalian Chk1 appears to be important for integrating DNA-damage repair with cell-cycle arrest. RESULTS: We have identified structural homologs of yeast Chk1 in human and mouse. Chk1(Hu/Mo) has protein kinase activity and is expressed in the testis. Chk1 accumulates in late zygotene and pachytene spermatocytes and is present along synapsed meiotic chromosomes. Chk1 localizes along the unsynapsed axes of X and Y chromosomes in pachytene spermatocytes. The association of Chk1 with meiotic chromosomes and levels of Chk1 protein depend upon a functional Atm gene product, but Chk1 is not dependent upon p53 for meiosis I functions. Mapping of CHK1 to human chromosomes indicates that the gene is located at 11q22-23, a region marked by frequent deletions and loss of heterozygosity in human tumors. CONCLUSIONS: The Atm-dependent presence of Chk1 in mouse cells and along meiotic chromosomes, and the late pachynema co-localization of Atr and Chk1 on the unsynapsed axes of the paired X and Y chromosomes, suggest that Chk1 acts as an integrator for Atm and Atr signals and may be involved in monitoring the processing of meiotic recombination. Furthermore, mapping of the CHK1 gene to a region of frequent loss of heterozygosity in human tumors at 11q22-23 indicates that the CHK1 gene is a candidate tumor suppressor gene.

MR1-restricted mucosal-associated invariant T (MAIT) cells respond to mycobacterial vaccination and infection in nonhuman primates.

Studies on mucosal-associated invariant T cells (MAITs) in nonhuman primates (NHP), a physiologically relevant model of human immunity, are handicapped due to a lack of macaque MAIT-specific reagents. Here we show that while MR1 ligand-contact residues are conserved between human and multiple NHP species, three T-cell receptor contact-residue mutations in NHP MR1 diminish binding of human MR1 tetramers to macaque MAITs. Construction of naturally loaded macaque MR1 tetramers facilitated identification and characterization of macaque MR1-binding ligands and MAITs, both of which mirrored their human counterparts. Using the macaque MR1 tetramer we show that NHP MAITs activated in vivo in response to both Bacillus Calmette-Guerin vaccination and Mycobacterium tuberculosis infection. These results demonstrate that NHP and human MR1 and MAITs function analogously, and establish a preclinical animal model to test MAIT-targeted vaccines and therapeutics for human infectious and autoimmune disease.

A new polymorphic determinant on HLA-DQ molecules.

In man, the immune response genes are located within the HLA-D/DR region, and the gene products, the Ia antigens, are expressed on B lymphocytes, monocytes, and a percentage of null cells and activated T lymphocytes. We recently identified a human Ia antigen, K19, which appeared to be limited in its expression to B lymphocytes, and to be preferentially expressed on the more mature cells within this population. This work was facilitated by a monoclonal antibody. HK-19, which recognized a monomorphic determinant of this Ia molecule. We now report the characterization of a second monoclonal antibody, HK-13, which recognized the same molecule as HK-19, but only on cells from some individuals. The greater affinity of HK-13 allowed more complete characterization of the K19/K13 molecule. This characterization included cytofluorography, two-dimensional gel electrophoresis, tryptic peptide mapping, and partial N-terminal amino acid sequencing, and indicated that K19 and K13 were epitopes on HLA-DQ (DC) molecules. The pattern of reactivity of HK-13 on a panel of typing cells did not correlate with any of the known HLA-DQ polymorphic determinants. Thus, HK-13 is a new polymorphic determinant of the HLA-DQ series.

Primary and secondary prostatic adenocarcinoma of the urinary bladder.

Urinary bladder involvement by prostatic adenocarcinoma (PAC) is not well characterized in the literature. Fifteen consecutive cases of PAC diagnosed in the urinary bladder over a period of 10 years were reviewed. All bladder and prostate slides from each patient were evaluated. Eleven patients (group A) had synchronous PAC in the prostate. In these patients, bladder PAC occurred 2 to 11 years after the initial diagnosis of PAC in the prostate and tended to have a higher Gleason score than the original prostatic PAC. Four cases of bladder PAC in group A had areas with features of urothelial carcinoma, with focal positive immunoreactivity for thrombomodulin in 2 cases. Two patients (group B) had undergone radical prostatectomy for PAC 15 years earlier. The lesions in the urinary bladder in both cases showed histopathologic features similar to those seen in the previous prostatic malignancies. Two patients (group C) had histories of previously resected urothelial carcinoma. Bladder PAC was diagnosed at routine follow-up, and repeated prostate biopsy up to 2 years after the diagnosis of bladder PAC showed no evidence of prostatic PAC. PAC in the urinary bladder may be either primary or secondary. Secondary PAC is usually associated with high-grade and high-stage carcinoma in the prostate and may mimic transitional cell carcinoma. Primary bladder lesions may or may not be associated with a history of PAC in the prostate. The prognosis of patients with the primary carcinoma is favorable. HUM PATHOL 32:434-440.

An unusual coronary vein lesion thrombus with calcification.

In this article, a patient with chronic renal failure and probable secondary hyperparathyroidism is described; calcification of a variety of cardiac and other structures complicated the latter. Calcification of an occlusive thrombus in the great cardiac vein and coronary sinus was identified by echocardiography, although not initially appreciated as such. The echocardiographic similarity of this lesion to mitral annular calcification is discussed.

Histologic estimation of coronary artery stenoses: reproducibility and the effect of training.

Histologic estimation of coronary artery stenoses (CAS) provides the 'gold-standard' for clinicopathologic correlations and medicolegal investigations, yet little evidence supports histology as a reproducible diagnostic measure, and none addresses the effect of training on its use. To study these questions, 20 randomly selected Movat-stained coronary artery cross-sections were reviewed 3 times, at 3-month intervals, by six clinical pathologists (CPs), six pathology residents (Res), seven anatomic pathologists (APs), and two cardiovascular pathologists (CVPs). Before the third iteration, a guide to CAS assessment with illustrations was provided. Inter- and intraobserver reproducibility were determined using interclass correlation coefficients (ICC) (0.40-0.75 = fair-good; > or = 0.76 = excellent agreement beyond chance). Surprisingly, all study groups had excellent interobserver reproducibility. Before training, at Time 1, the scores were CPs, 0.77; Res, 0.89; APs, 0.93; and CVPs, 0.93. After training, at Time 3, the results were CPs, 0.81; Res, 0.91; APs, 0.86; and CVPs, 0.88. Intraobserver reproducibility for CPs overall was good (ICC, 0.74), and excellent for Res, APs, and CVPs (0.89, 0.94, and 0.97, respectively). In conclusion, statistical analysis failed to demonstrate any significant effect of training or experience on observer reproducibility.

The histologic estimation of coronary artery stenoses: accuracy and the effect of lumen shape.

The authors have recently investigated the histologic estimation of coronary artery stenoses (CAS) to determine its reproducibility and the effect of training on reproducibility. The present study extends this work, examining the accuracy, the sensitivity, and the specificity of the estimation of CAS. Further, the effect of one histologic variable (i.e., arterial lumen shape) on the evaluation of CAS is examined. As described previously, 20 randomly selected Movat-stained coronary artery cross-sections were reviewed three times, at 3-month intervals, by six clinical pathologists (CPs), six pathology residents (Res), seven anatomic pathologists (APs), and two cardiovascular pathologists (CVPs). Before the third iteration, training in CAS assessment was provided. In the present study, for comparison with observer estimates, image analysis was used to establish the actual percent CAS and determine observer accuracy. The results of this study showed, paradoxically, that greater experience did not correlate with greater accuracy: The CPs consistently had the highest accuracy scores and the CVPs consistently had the lowest. Training, however, improved the accuracy scores of all groups. Stenotic arterial cross-sections with residual lumens showing concentric or eccentric polymorphous shapes were consistently underestimated compared to image analysis, while lumens with a eccentric slitlike shape were consistently overestimated.

Experimental spinal cord injury: imaging the acute lesion.

In order to obtain high resolution images of fixed excised rat spinal cords we have developed a technique using a 6-mm bore, two-turn saddle coil, with a usable imaging length of approximately 4 cm. MR imaging is performed on a prototype 31-cm bore, 1.9-T system with a 1.5-mm section thickness and 7.6-mm field of view.

Predictive value of extent and grade of ductal carcinoma in situ in radiologically guided core biopsy for the status of margins in lumpectomy specimens.

AIMS: The correlation between the extent and grade of ductal carcinoma in situ (DCIS) in a core needle biopsy of breast, and the presence of an extensive intraductal carcinoma component (EIC) or positive resection margins in a subsequent mastectomy, has not been adequately addressed in the literature. MATERIALS AND METHODS: Seventy-eight core needle biopsies with mammography and mastectomy correlation (27 total mastectomies, 51 lumpectomies) were reviewed. The extent and grade of DCIS in the biopsies were determined and compared with the mammographic findings and the status of the EIC and margins in subsequent mastectomy specimens. RESULTS: Twenty-four cases of core biopsies with at least three foci of low-grade DCIS or at least two foci of high grade DCIS (group I) corresponded in large part to cases of mastectomy with a positive EIC (20/23 cases, or predictive value of 87%). Nine of 15 cases of lumpectomy in this group were associated with margins positive for or close to (less than 0.1 cm from) carcinoma. Thirty-three cases of core biopsies with one or two foci of low-grade DCIS or one focus of high-grade DCIS (group II) were associated with mastectomies with a limited extent of DCIS. Only four of 22 lumpectomy specimens in this group had margins positive for or close to carcinoma. Twenty-one cases of core biopsies without DCIS (group III) represented all five mastectomy specimens without DCIS, and 16 mastectomies with DCIS and negative EIC. None of the 14 cases of lumpectomy in this group had margins positive for carcinoma. The predictive value for EIC status may be even higher if mammographic findings are used in cases with a low number of foci (two foci of low-grade DCIS or one focus of high-grade DCIS in short biopsy cores). CONCLUSIONS: There was a good correlation between the extent and grade of DCIS in core biopsies and the status of EIC in subsequent mastectomy specimens. Core needle biopsies with at least three foci of low-grade DCIS or at least two foci of high-grade DCIS are associated with a greater likelihood of positive or close margins in subsequent lumpectomies. Core biopsies without DCIS are associated with a greater likelihood of negative margins in subsequent lumpectomies.

Comparison of methods for extracting linear solvent strength gradient parameters from gradient chromatographic data.

The linear solvent strength (LSS) theory of gradient elution is useful in the optimization of separations in high-performance liquid chromatography. While the fundamental parameters of this theory are defined in terms of isocratic behavior, gradient operation has been used previously to estimate those parameters to allow rapid optimization of the separation. In this study, various methods of extracting the LSS parameters from gradient retention data were examined. Sets of synthetic retention data were calculated directly from the equations of the LSS theory. When realistic experimental uncertainties were incorporated into these data sets, the LSS parameters used to generate the synthetic data were not recovered accurately unless special precautions were taken. For large molecules, an approximate LSS expression could be used to determine the solvent strength parameter with an error of less than 13%, which is comparable to or better than those for the other methods evaluated.

Comparison of selected retention models in reversed-phase liquid chromatography.

Retention models are usually compared by how well the model equation fits retention data for one solute taken over a range of mobile phase compositions. Even when retention data for multiple solutes are used, the quality of the fit is often judged by the statistical goodness-of-fit alone. This study compared four different RPLC retention models, encompassing three distinct mathematical forms. Each model was fit to the retention data of multiple solutes and the sets of best-fit parameters were examined in terms of the underlying physico-chemical assumptions of the models. Next, for the linear and quadratic models, some of the model parameters were calculated a priori and the rest of the model parameters were then obtained in subsequent fittings. The sets of best-fit parameters obtained in this manner were more consistent with the underlying assumptions of these models than were the sets of parameters obtained entirely through regressions to the experimental data. Thus, the extraction of parameters by fitting a model to the retention data of a single solute may result in unreliable values for those parameters, even in the case of a fit that would be considered good when judged by conventional statistical criteria. That is, although parameters extracted in such a fashion may be suitable for optimization or similar uses, they may not be suitable for determining the appropriateness of the underlying assumptions of retention models.

Modeling the effect of solvation on solute retention in reversed-phase liquid chromatography.

The retention of a homologous series of alkylbenzenes was determined on octyl and octadecyl reversed-phase columns in several polar organic liquids. Free energies of transfer were calculated by the SM5.0R classical solvation model for each organic liquid tested and for several alkanes. The relationships between the measured retention factors and the calculated free energies of transfer were then investigated. Although the natural logarithms of the retention factor and the calculated free energies of transfer were linearly correlated, the obtained free energies of transfer of the solutes did not completely explain the retention behavior of the solutes. Nonetheless, even in these pure organic liquids, the energetics of RPLC retention behaved very similarly to those of partitioning.

Magnetic resonance imaging observations of blood-brain-barrier permeability in an animal model of brain injury.

RATIONALE AND OBJECTIVES: Knowledge of the in vivo relaxivity of paramagnetic contrast agents is important in the accurate measurement of the permeability of the blood-brain barrier (BBB). This study was aimed at developing an animal model for the magnetic resonance (MR) imaging investigation of injuries to the BBB. METHODS: MR imaging (1.9 T) was performed in 18 rats with acute, stable injuries to the brain caused by freezing. After injection of gadodiamide (0.05-0.20 mmol/kg), estimates were made of BBB permeability, leakage space, and relaxivity (also measured in saline). RESULTS: The BBB was always disrupted at the injured site (permeability = 0.038 min-1 +/- 0.0006). The central area of necrosis and the periphery of edema showed substantial differences in leakage space and relaxivity. The relaxivity of gadodiamide was much greater at the injured site than in saline. CONCLUSION: The in vivo relaxivity at a site of pathologic change in the brain may be substantially greater than that measured in aquo.

Comparison of new clinical and scintigraphic algorithms for the diagnosis of pulmonary embolism.

Since the publication of the modified Prospective Investigation of Pulmonary Embolism Diagnosis (PIOPED) criteria for the diagnosis of pulmonary embolism (PE), new clinical and scintigraphic diagnostic algorithms (the McMaster clinical criteria, the PisaPED simplified scintigraphic grading and the Miettinen logistic regression analysis) have been reported although the results have not been reproduced in other sites. Ventilation-perfusion lung scintigraphy was performed in 238 consecutive patients with a provisional diagnosis of PE. Scans were reported as normal/very low, low, intermediate or high probability for PE using standardized criteria. Each patient received a clinical grading of probability of PE as low, moderate or high using the McMaster clinical criteria. Using the PisaPED criteria (an alternate simplified scintigraphic grading system using the perfusion scan alone) each scan was also graded as normal/near normal, abnormal but not PE, or abnormal and PE. Using the logistic regression algorithm of Miettinen each scan received a numerical probability of PE. Frequencies for differing levels of probability of PE varied widely between the various algorithms. Cross tabulations revealed correlation of the standardized criteria with the Miettinen grading but not with the McMaster or the PisaPED gradings. We were unable to reproduce similar results using the McMaster clinical grading or the PisaPED simplified scintigraphic grading although the Miettinen logistic regression formula gave comparable results. New algorithms are not automatically transferable to new environments.

Immunohistochemical study of papillary thyroid carcinoma and possible papillary thyroid carcinoma-related benign thyroid nodules.

Recent immunohistochemical studies have identified different antisera that have various degrees of sensitivity and specificity for papillary thyroid carcinoma (PTC). In this study, we performed immunostaining for CK, EMA, HBME, CD57 and CD15 in PTC, and benign thyroid nodular lesions to compare the sensitivity and the specificity of these antisera for PTC. In addition, we studied the patterns of immunostaining of these antisera in benign nodular thyroid lesions displaying a fine chromatin pattern, foci of cells with nuclear grooves, and optically clear nuclei. Fifty-five PTC (composed of 30 papillary variants and 25 follicular variants), 5 follicular carcinomas, 30 follicular adenomas, and 20 thyroid nodular lesions (5 papillary variants and 15 follicular variants) were submitted for immunostaining with CK, EMA, HBME, CD57, and CD15. CK and HBME showed the highest sensitivity and specificity for PTC when an arbitrary cutoff of more than 10% positive cells was considered as positive diagnostic immunostaining for these sera. The other antisera were less sensitive and less specific. One case of PTC showed negative HBME but positive CD15, whereas three papillary variants and two follicular variants of benign thyroid nodules revealed a positive diagnostic HBME immunostaining for PTC and negative CK immunostaining. Any combination of positive diagnostic immunostaining with CK+ HBME, CK+ CD57 or CK+ CD15 has a sensitivity of 95% and specificity of 90% for PTC. Thyroid nodules with a diffuse or focal fine chromatin pattern and focal areas with nuclear grooves or optically clear nuclei displayed immunoreactivity ranging from 0% to 50% of cells. Three of five follicular carcinomas showed negative reactivity for HBME, CD57, and CD15. A combination of immunostaining with CK, HBME and CD57 (or CD15) is a sensitive and specific test for PTC. This panel can be used to rule out thyroid nodules posing a diagnostic problem with PTC. Follicular adenoma and nodules of the thyroid, with a fine chromatin pattern and focal nuclear grooves or optically clear nuclei, displayed an intermediate range of reactivity between reactive thyroid tissue and PTC.