Determining the impact of increasing standardized ileal digestible lysine for primiparous and multiparous sows during lactation.

Two experiments were conducted to evaluate the effects of increasing dietary SID Lys in lactation on sow and litter performance. In Exp. 1, a total of 111 primiparous sows (Line 241; DNA Genetics, Columbus, NE) were allotted to 1 of 4 dietary treatments on d 110 of gestation. Dietary treatments included increasing dietary standardized ileal digestible (SID) Lys (0.80, 0.95, 1.10, and 1.25%). During lactation, there were no differences in ADFI or sow BW at weaning (d 21), resulting in no differences in BW loss. However, backfat loss during lactation decreased (linear, P = 0.046) as SID Lys increased. There were no differences in litter weaning weight, litter gain from d 2 to weaning, percentage of females bred by d 7 after weaning, d 30 conception rate, farrowing rate or subsequent litter characteristics. In Exp. 2, a total of 710 mixed parity sows (Line 241; DNA Genetics) were allotted to 1 of 4 dietary treatments at d 112 of gestation. Dietary treatments included increasing SID Lys (0.75, 0.90, 1.05, and 1.20%). Sow BW at weaning increased (quadratic, P = 0.046), and sow BW loss from post-farrow to weaning or d 112 to weaning decreased (quadratic, P ≤ 0.01) as SID Lys increased. Sow backfat loss increased (linear, P = 0.028) as SID Lys increased. Conversely, longissimus muscle depth loss decreased (linear, P = 0.002) as SID Lys increased. Percentage of females bred by d 7 after weaning increased (linear, P = 0.047) as SID Lys increased in parity 1 sows, with no difference in parity 2 or 3+ sows. Litter weight at d 17 and litter gain from d 2 to 17 increased (quadratic, P = 0.01) up to 1.05% SID Lys with no improvement thereafter. For subsequent litter characteristics, there were no differences in total born, percentage born alive, stillborn, or mummies. In conclusion, our results suggest that increasing dietary SID Lys can reduce sow protein loss in lactation. The optimal level of dietary SID Lys required by the sow may vary based on response criteria and parity.

Impaired bronchial healing after lung donation from non-heart-beating donors.

BACKGROUND: Bronchial viability after lung transplantation remains a concern. Modern preservation methods, surgical technique, and limited cold ischemic periods have decreased the frequency of bronchial complications. However, lungs procured from non-heart-beating donors are subjected to a mandatory period of warm ischemia. We investigated bronchial healing in a porcine survival model of left lung transplantation using organ procurement from non-heart-beating donors after a 60-minute period of warm ischemia. METHODS: Fourteen adult domestic swine underwent left lung transplantation. All lungs were preserved with cold Euro-Collins flush and stored inflated at 4 degrees C. Control lungs (n = 5) were flushed, harvested, and stored for 2 hours before implantation. Experimental lungs (n = 9) were procured from non-heart-beating donors. These lungs were subjected to 60 minutes of warm ischemia before flush and harvest, followed by 2 hours of cold storage before implantation. After 21 days of immunosuppression with prednisone, azathioprine, and cyclosporine, pulmonary function was assessed. Bronchial viability was evaluated with bronchoscopy and, at autopsy, followed by histologic analysis. RESULTS: Implantation time did not differ significantly between the control group and the experimental group (59.6 +/- 2.1 versus 64.4 +/- 2.9 minutes, p = 0.24). Control swine exhibited no evidence of ischemic injury to the donor bronchus. In contrast, six of nine lungs procured from non-heart-beating donors showed evidence of ischemic bronchial injury (p = 0.031 versus control). Findings ranged from hypovascular edematous mucosa to necrosis and sloughing of the mucosa throughout the entire donor bronchial tree. The remaining three non-heart-beating donor lungs exhibited normal lung function and bronchial healing. CONCLUSIONS: We conclude that 60 minutes of warm ischemia for lungs procured from non-heart-beating donors results in impaired bronchial viability with current preservation techniques. Thirty minutes of warm ischemia may be the acceptable limit for lung procurement from non-heart-beating organ donors.

Automated immunohistochemical assay for estrogen receptor status in breast cancer using monoclonal antibody CC4-5 on the Ventana ES.

Determination of breast cancer estrogen receptor (ER) status as a predictor of tumor response to adjuvant endocrine therapy remains a mainstay of breast cancer management. Recent second generation anti-ER antibodies and new epitope retrieval methods have produced paraffin-based immunohistochemical results that correlate closely with the dextran-coated charcoal (DCC) assay and appear to represent a superior method of ER assay. The authors determined the ER status of 103 invasive breast cancers by paraffin-based, automated immunohistochemistry on the Ventana ES using a new monoclonal antibody, CC4-5, and compared the results to those of parallel DCC biochemical analysis and manual immunohistochemical analysis using anti-ER monoclonal antibody ER1D5. The specificity of the CC4-5 antibody for ER protein was confirmed by Western blot analysis. Sixty of 103 cases were positive for ER by CC4-5 automated immunohistochemistry. With a ligand binding assay threshold value of 20 fmol/mg protein, there were 50 positive cases by biochemical assay. The biochemical results corresponded to an 88% rate of agreement with automated CC4-5 staining. Analysis of discordant cases revealed that the majority of CC4-5 immunopositive only cases (8 of 11) were strongly positive, stroma rich tumors, suggesting that corresponding biochemical measurements were diluted by non representative stromal tissue. There was only one immunonegative, biochemically positive case (27 fmol/mg protein). Semiquantitation of CC4-5 staining using percent positive tumor cells or weighted average staining intensity (HSCORE) showed moderate to good correlation with quantitative DCC results (r = 0.64 and 0.62, P < .0001). ER1D5 was not suitable for use on the Ventana ES, most likely due to temperature constraints of the instrument. By manual ER1D5 staining, 40 of 79 examined cases were positive corresponding to a 99% rate of agreement with automated CC4-5 staining. Semiquantitation of ER1D5 staining by percent positive tumor cells and weighted average staining intensity (HSCORE) showed excellent correlation with semiquantitation of automated CC4-5 results (r = 0.90 and 0.88, P < .0001). Automated immunohistochemistry using the Ventana ES and monoclonal antibody CC4-5 is a reliable method for determining breast cancer ER status. As with other immunohistochemical methods, direct correlation with morphology precludes errors due to tissue sampling, allowing for accurate analysis of stroma-rich or partially necrotic tumors and small neoplasms that otherwise would yield insufficient tumor tissue for biochemical analysis.

Cyclin D1 gene expression in human cervical neoplasia.

Amplification of chromosome 11q13 leads to overexpression of a G1 cyclin gene, cyclin D1 (PRAD-1, CCND-1), in many non-cervical human carcinomas. Homology between cyclin D1 and human papillomavirus oncoprotein E7 binding sites for the retinoblastoma tumor-suppressor protein suggests that human papillomavirus oncoproteins cyclin D1, and other cell cycle regulatory proteins may act through a common mechanism in the pathogenesis of human cervical squamous cell carcinoma. We have examined 48 cases of cervical neoplasia by RNA-mRNA in situ hybridization for human papillomavirus mRNA and cyclin D1 mRNA and by immunohistochemistry for cyclin D1 protein expression. Hybridization demonstrated human papillomavirus RNA in all 48 cases with types 6, 16, or 18 in 2, 26, and 20 cases, respectively. Immunohistochemical detection using anti-cyclin D1 rabbit polyclonal antibody 19 demonstrated appropriate cyclin D1 expression at constitutive low levels in normal squamous epithelium and low-grade intraepithelial lesions. Immunohistochemical staining failed to demonstrate significant protein expression in any of the high-grade or invasive lesions. In contrast to the immunohistochemical results, in situ hybridization demonstrated cyclin D1 mRNA overexpression in three of five cases of low-grade squamous intraepithelial lesion, one of eight cases of high-grade squamous intraepithelial lesion, 14 of 18 cases of invasive squamous cell carcinoma, two of five cases of adenocarcinoma in situ, one of seven cases of invasive adenocarcinoma, and two of five cases of small cell undifferentiated carcinoma. Transcriptional activation of cyclin D1 can occur in vivo in human papillomavirus-associated invasive cervical carcinoma, but it does not seem to result in a steady state, increased level of cyclin D1 protein expression. These data indicate a limited role for cyclin D1 protein in the pathogenesis of human papillomavirus-associated invasive cervical squamous carcinoma. They support a model in which human papillomavirus proteins can circumvent cellular requirements for cyclin D1 in human cervical neoplasia.

Cytologic diagnosis of ovarian tumors: factors influencing accuracy in previously undiagnosed cases.

OBJECTIVES: Cytologic diagnosis of ovarian masses by needle aspiration techniques remains controversial. This review proposes to define the accuracy of the technique, report complications, and relate clinical situations in which the technique was used. STUDY DESIGN: In a retrospective review all patients undergoing cytologic aspiration biopsy diagnosis of ovarian masses at the University of Virginia Health Sciences Center from 1986 through 1993 were identified, and 74 women with corresponding histologic material were used. Clinical data were abstracted and all cytologic and pathologic material was reviewed. RESULTS: The overall sensitivity of the 74 aspiration biopsies to predict the histologic diagnosis of malignancy was 78%; specificity was 92%. Two patients had complications, one necessitating operative intervention. Correct diagnoses were influenced by menopausal status, patient age, sample type, aspiration method, and cytologic quality. CONCLUSIONS: The cytologic diagnoses of ovarian tumors, while quite specific, lack the sensitivity for general application. Use of this diagnostic technique must remain individualized, and the factors that influence the accuracy of the technique must be kept in mind. There remains the need for standardization of reporting fine needle aspiration results.

Lymphocyte predominance Hodgkin's disease. Evidence for a kappa light chain-restricted monotypic B-cell neoplasm.

Over the last decade, it has been noted that nodular lymphocytic and/or histiocytic predominance Hodgkin's disease (NLPHD) has clinical, histological, and immunophenotypical differences from classical Hodgkin's disease, but it is not clear whether NLPHD represents a B-cell neoplasm or merely an unusual B-lineage reactive condition. We evaluated 36 cases of LPHD (31 nodular, 5 diffuse) for evidence of B-cell clonality by immunohistochemistry for light-chain protein using polyclonal antibodies and microwave antigen retrieval, and by a highly sensitive in situ hybridization technique for light-chain mRNA using 3H-labeled antisense RNA probes. We found monotypic light-chain restriction for kappa protein in 36% of cases, with no clear predominance for either light-chain protein in the other cases. By in situ hybridization, 80% of the evaluable cases showed clear evidence of light-chain monotypsim, with 96% of cases monotypic for kappa mRNA and one case monotypic for lambda mRNA. In virtually all of these cases, the L&H cells were found to be monotypic, consistent with monoclonality. In about one-half of these cases, a small lymphocytic component was also found to be monotypic. Our data support the hypothesis that NLPHD and its rare diffuse variant represent a monotypic B-cell neoplasm, almost always of kappa light-chain type. NLPHD represents a neoplasm distinct from classical Hodgkin's disease.

In situ hybridization detection of cyclin D1 mRNA in centrocytic/mantle cell lymphoma.

BACKGROUND: Centrocytic/mantle cell lymphoma (MCL) is characterized by a specific chromosomal translocation, t(11;14)(q13;q32), which leads to deregulated expression of the G1 cyclin, cyclin D1 (PRAD1, CCND1, BCL1). Cyclin D1 overexpression has been demonstrated in MCL at the mRNA level by Northern blotting and at the protein level by both Western blotting and immunoperoxidase staining. PATIENTS AND METHODS: To assess the utility of in situ hybridization (ISH) to detect cyclin D1 mRNA expression in formalin-fixed, paraffin embedded tissue, five MCL specimens from three patients and two cases of B-cell small lymphocytic lymphoma (B-SLL) were studied. BCL1 major translocation cluster gene rearrangements had been previously documented in two MCL patients; the other MCL and the two B-SLL, showed no detectable BCL1 or cyclin D1 rearrangements. RESULTS: ISH was performed using anti-sense 3H-labeled RNA probes for the cyclin D1 3' untranslated region (pPL7) and partial cyclin D1 cDNA (pPL8). ISH experiments using an anti-sense actin RNA probe demonstrated adequate RNA preservation in all cases. Each of five specimens of MCL demonstrated increased cyclin D1 mRNA. In contrast, neither of the two cases of B-SLL demonstrated detectable levels. CONCLUSIONS: Overexpression of cyclin D1 mRNA can be detected in MCL by ISH using formalin fixed paraffin embedded tissue. The ISH technique may be useful in diagnosing and classifying low-grade B-cell lymphomas and should be applicable to the study of cyclin D1 mRNA expression in a broad spectrum of lymphoid proliferations and solid tumors.

Lymphoproliferative disorder of granular lymphocytes: nine cases including one with features of CD56 (NKH1)-positive aggressive natural killer cell lymphoma.

Twelve cases of large granular lymphocytosis were examined by flow cytometry and Southern blot analysis to correlate immunophenotypic and molecular genetic markers with clinical features. Nine cases fulfilled clinical criteria for the lymphoproliferative disorder of granular lymphocytes, and eight of these demonstrated the molecular features of T-cell-type lymphoproliferative disorder of granular lymphocytes including surface expression of CD3, expression of one or more natural killer (NK)-cell antigens (CD11b, CD16, or CD57), and clonal rearrangement of both the T-cell receptor beta- and gamma-chain-joining genes. One of these cases demonstrated coexisting clonal rearrangement of the immunoglobulin heavy-chain-joining genes, but none demonstrated kappa-light-chain-joining gene rearrangement. The eight T-cell lymphoproliferative disorder of granular lymphocytes cases all lacked expression of the NK antigen CD56 (NKH1). In contrast, the other case of lymphoproliferative disorder of granular lymphocytes rapidly evolved into an aggressive NK-cell lymphoma which did not express CD3, did express CD56, had germline T-cell receptor gene configurations, and had multiple clonal chromosomal abnormalities. This case demonstrated nasal cavity and cutaneous tumor infiltrates consistent with previously described CD3-negative, CD56-positive NK-cell lymphoma of the upper aerodigestive tract. Three cases of transient large granular lymphocytosis demonstrated germline T-cell receptor gene configurations. This study demonstrates the usefulness of Southern blot analysis and flow cytometry in characterizing proliferations of large granular lymphocytes. The transformation of a single case into an aggressive NK-cell lymphoma with blastic morphology and tissue infiltration was associated with a fatal outcome.

Low-grade fibromyxoid sarcoma: case report and immunohistochemical study.

A case is presented of low-grade fibromyxoid sarcoma involving the arm of a 52-year-old man. Low-grade fibromyxoid sarcoma is a recently described neoplasm of the deep and subcutaneous soft tissue which demonstrates a spectrum of histologic images. The current case demonstrated the typical patterns of intermixed, sweeping bands of fibrous and myxoid tissue, homogeneous foci of fibrous and myxoid tissue, focal areas of storiforming, and concentric perivascular cuffs of slender spindle cells, all lacking the nuclear anaplasia, mitotic activity, and necrosis generally associated with sarcoma. Immunohistochemical analysis performed on paraffin-embedded sections demonstrated strong labeling of the tumor cells by anti-CD34 antibody, moderate labeling for vimentin, and rare, focal positivity for muscle-specific actin. Tumor cells were negative for markers of epithelial, muscular, neural, histiocytic, melanocytic, and vascular differentiation. The constellation of histopathologic features described in this and previous reports is characteristic of low-grade fibromyxoid sarcoma. Based on this case, it appears that the immunohistochemical features of low-grade fibromyxoid sarcoma can help to exclude many cutaneous and deep soft tissue tumors from the differential diagnosis. The findings support the interpretation that the neoplasm is essentially fibroblastic in nature.

Lobular capillary hemangioma. An immunohistochemical study including steroid hormone receptor status.

Twenty-one lobular capillary hemangiomas (LCH), including lesions from six pregnant patients, were examined by immunohistochemical analysis. Antibodies to estrogen and progesterone receptor proteins were used to determine whether these steroid hormones play a direct role in LCH development and growth. All 21 LCHs were negative for both receptor proteins. Contrary to the association of LCH with pregnancy and oral contraceptive use, the absence of these steroid receptors strongly suggests that estrogen or progesterone are not directly involved in the formation of these lesions. All 21 LCHs were stained with Ulex europaeus lectin and with a panel of antibodies directed against cytokeratin, vimentin, Factor VIII, collagen Type IV, and muscle-specific actin. Endothelial cells in LCH, both in cellular proliferations with poorly formed lumens and in well-formed capillaries, were labeled by Factor VIII, Ulex europaeus lectin, and vimentin. A population of concentrically arranged, perivascular spindle-shaped cells was highlighted by positive staining for muscle-specific actin, collagen Type IV, and vimentin. The spindled cells were associated with both well-developed and immature vessels in all lesions. The appearance, immunophenotype, and location of these cells is consistent with a pericytic origin. Although the intimate association of both endothelial cells and pericytes suggests a reactive, as opposed to neoplastic, origin, other benign vascular tumors traditionally considered neoplastic have a similar bicellular composition. Accordingly, the findings neither support nor refute a neoplastic origin for LCH.

Spindle cell mural nodules in cystic ovarian mucinous tumors. A clinicopathologic and immunohistochemical study of five cases.

We studied five cystic ovarian mucinous tumors with spindle cell mural nodules to define their histologic and immunohistochemical properties. Three of these mural nodules consisted of carcinomatous nests surrounded by highly pleomorphic polygonal and spindle cells. There were transition zones between the pleomorphic cells and the cell nests. In all three cases, both cell populations coexpressed cytokeratin and vimentin, suggesting a diagnosis of anaplastic carcinoma. A fourth nodule consisted of moderately differentiated adenocarcinoma embedded in prominent, cytologically uniform spindle cells. These cells were histologically distinct from the carcinoma; there were no zones of transition. The carcinoma was strongly positive for cytokeratin but only weakly positive for vimentin; the spindle cells expressed vimentin but not cytokeratin. We diagnosed this lesion as carcinoma with a reactive spindle cell stroma. A fifth mural nodule was composed entirely of interlacing fascicles of uniform spindle cells that were negative for cytokeratin but positive for vimentin, muscle-specific actin, and desmin; these findings support a diagnosis of leiomyoma. Two of the four patients with malignant nodules died of disease; the rest are alive and disease-free after follow-up intervals ranging from 1 to 4 years. This study demonstrates the usefulness of immunohistochemistry in distinguishing variant forms of spindle cell mural nodules in cystic ovarian mucinous tumors. It further suggests that malignant nodules do not necessarily carry a poor prognosis.

White sponge nevus.

A case is presented of white sponge nevus involving the vaginal, labial, and oral mucosae of a 34-year-old woman. White sponge nevus is a rare, benign, autosomal dominant leukokeratosis that predominantly affects the oral mucosa. Less frequently, it affects extraoral sites including the vulvovaginal mucosa. In this case, histopathologic study of vulvovaginal lesions, subsequent examination of extragenital mucosae, and inquiry into the family history led to the correct diagnosis. The genetics, clinical appearance, and histopathology of white sponge nevus are discussed in relation to the differential diagnosis of oral and vaginal leukokeratoses.

Polarity of neutral glycolipids, gangliosides, and sulfated lipids in MDCK epithelial cells.

Confluent monolayers of MDCK (Madin-Darby canine kidney) cells provide a widely used model system for studying epithelial cell polarity. We determined the polarity of epithelial cell plasma membrane glycolipids and sulfated lipids by analyzing the lipids released from both sides of monolayers of metabolically labeled MDCK cells. These lipids were released either as endogenously shed material or in budding viruses. All of the glycolipids were detected in both the apical and basolateral domains of the plasma membrane. However, galactosylceramide was more basally oriented than any of the other glycolipids; thus, the ratio of glucosylceramide to galactosylceramide was more than twice as great in the apical domain as in the basolateral domain. A sulfated sterol, which comigrated with cholesterol sulfate, was released in a more basally polarized manner than any of the glycolipids. These results indicate the presence of mechanisms which can produce different degrees of polarity for specific lipids in polarized epithelial cells.

Polarity of the Forssman glycolipid in MDCK epithelial cells.

To determine whether epithelial plasma membrane glycolipids are polarized in a manner analogous to membrane proteins, MDCK cells grown on permeable filters were analyzed for the expression of Forssman ceramide pentasaccharide, the major neutral glycolipid in these cells. In contrast to a recent report which described exclusive apical localization of the Forssman glycolipid (Hansson, G.C., Simons, K. and Van Meer, G. (1986) EMBO J. 5, 483-489), immunofluorescence and immunoelectron microscopic staining revealed the Forssman glycolipid on both the apical and basolateral surfaces of polarized cells. Immunoblots indicated that the Forssman antigen was detectable only on glycolipids and not on proteins. Analysis of metabolically labeled glycolipids released into the apical and basal culture medium, either as shed membrane vesicles or in budding viruses, also demonstrated the presence of the Forssman glycolipid on both apical and basolateral membranes of polarized cells. Quantitation of the released glycolipid indicated that the Forssman glycolipid was concentrated in the apical membrane. These results are consistent with previous reports which described quantitative enrichment of glycolipids in the apical domain of several epithelia.

On tight junction structure: Forssman glycolipid does not flow between MDCK cells in an intact epithelial monolayer.

One model of tight junction structure suggests that lipids might flow from cell to cell within shared exoplasmic membrane leaflets. We tested this proposal by co-culturing two clones of MDCK epithelial cells, which differed in their content of Forssman glycolipid, and then staining by immunofluorescence with rabbit anti-Forssman Ig. In co-cultures grown on glass cover slips and on nitrocellulose filters, positive Forssman staining was restricted to sharply demarcated clusters of cells formed by the Forssman-positive clone. Integrity of tight junctions between the two clones was indicated on cover slips by the presence of individual domes (hemicysts) composed of both clones and on filters by the generation of transepithelial potential differences. These results suggest that glycolipids in the exoplasmic leaflet of cells in a tight epithelium do not flow to adjacent cells.

Isolation and characterization of two types of MDCK epithelial cell clones based on glycosphingolipid pattern.

The Madin-Darby canine kidney (MDCK) epithelial cell line was shown previously to be heterogeneous with marked differences reported between low-passage (strain I) and high-passage (strain II) cultures (Richardson, J.C.W., Scalera, V. and Simmons, N.L. (1981) Biochim. Biophys. Acta 673, 26-36). This report describes major differences in the glycolipids of the two subpopulations of cells that comprise strain I and strain II cultures. The majority of strain II cells were strongly positive for the Forssman glycolipid antigen, while strain I cells were Forssman-deficient. Upon finding that strain I cells were contaminated with mycoplasma, we rescued Forssman-deficient cells from strain II using an anti-Forssman plus complement lysis procedure. Clones of surviving cells consisted of two distinct cell types. The first were Forssman-deficient, non-ciliated, spindle-shaped cells which generated negative (apical to basolateral) transepithelial potential differences. Clones of the second type were strongly Forssman-positive, ciliated, and formed island-shaped clusters of cuboidal cells. These latter clones generated positive potential differences and grew more slowly than the spindle-shaped clones. Spindle cells were enriched in fucolipids, while cuboidal cells contained higher levels of sulfated glycolipids. These two types of clones should provide excellent model systems in which to study the processing and polarity of glycolipids in epithelial cells.

Cosmic-ray tracks in plastics: the apollo helmet dosimetry experiment.

Counts of tracks from heavy cosmic-ray nuclei in helmets from Apollo missions 8 and 12 show variations caused by solar modulation of the galactic cosmic-ray flux. Specific estimates of the biological damage to certain nonreplaceable cells by track-forming particles during these space missions indicate that the fraction of deactivated cells could range from a lower limit of 3 x 10(-7) to an upper limit of 1.4 x 10(-4).