Mucolipidosis IV in an African American patient with new findings on electron microscopy.

PURPOSE: We report an unusual case of mucolipidosis IV in a patient of African ancestry, with intracytoplasmic inclusions of the corneal endothelium found on electron microscopy. METHOD: Clinical description with light and electron microscopy. RESULTS: We describe a case of mucolipidosis IV diagnosed in a patient of African ancestry after penetrating keratoplasty. Electron microscopic evaluation revealed intracytoplasmic inclusions in both the corneal epithelium and endothelium. CONCLUSION: The diagnosis of mucolipidosis in a patient of African ancestry is unusual, as this genetic disorder is found predominantly in individuals of Jewish descent. Corneal endothelial involvement in mucolipidosis IV has not previously been reported.

Histopathological features of ocular toxoplasmosis in the fetus and infant.

BACKGROUND: Ocular disease is a frequent manifestation of congenital Toxoplasma gondii infection. There are only limited data available in the literature concerning early stages of this disease in fetuses and infants. The purpose of our study was to characterize histopathological features in the eyes of 10 fetuses and 2 infants with congenital toxoplasmosis. METHODS: Fifteen eyes from 10 fetuses, 3 eyes from 2 premature infants, and both eyes from a 2-year-old child with congenital toxoplasmosis were examined by light microscopy. Immunohistochemical analysis to identify inflammatory cells and T gondii antigens was performed. The findings in infected eyes were compared with those of age-matched control eyes. RESULTS: Retinitis (10/18 eyes), retinal necrosis (4/18 eyes), disruption of the retinal pigment epithelium (12/18 eyes), and choroidal inflammation and congestion (15/18 eyes) were characteristic findings. Optic neuritis was present in 5 of 8 fetal eyes with associated optic nerve available for evaluation. An eye obtained from a 32-week-old fetus showed retinal rosettes at the edge of a scar. T cells predominated in retinal lesions and choroid. Parasites were identified by immunohistochemical analysis in 10 of 18 eyes. CONCLUSIONS: Ocular toxoplasmosis causes irreversible damage to the retina in utero. The fetus and infant mount inflammatory responses that may contribute to ocular damage. These findings have important implications for serological screening programs and in utero therapy.

Dimension-based visual attention modulates dual-judgment accuracy in Duncan's (1984) one- versus two-object report paradigm.

Four experiments, adapting the object-judgment paradigm developed by J. Duncan (1984), examined the relationship between object-based and domain-based mechanisms of visual attention. The experiments demonstrated a cross-domain cost, in terms of accuracy, when observers made dual color-form judgments to one or two overlapping objects presented briefly, relative to within-domain, dual-color and dual-form judgments. This domain-based selection effect was additive to an object-based effect, a cost of making dual judgments to separate objects, as reported by J. Duncan (1984). The pattern of object- and domain-based effects points to a capacity limitation in how multidimensional features are bound into a coherent object representation, consistent with the dimension-weighting account of H. J. Miller, D. Heller, and J. Ziegler (1995), which postulates that there is a limit to the total selection weight available to be allocated to an object's dimensions.

K-ras codon 12 mutations in Barrett's oesophagus and adenocarcinomas of the oesophagus and oesophagogastric junction.

BACKGROUND: Activation of the ras oncogene is commonly found in gastrointestinal tract cancers, but the role of ras in the development and progression of Barrett's oesophagus and associated cancers is uncertain. METHODS: The frequency of K-ras codon 12 point mutations was assessed in 52 paraffin-embedded tissues from 44 patients with oesophageal pathology. The specimens were classified pathologically as follows: adenocarcinoma of the oesophagus or oesophagogastric junction (n = 23), Barrett's high-grade dysplasia (n = 5), low-grade dysplasia (n = 14), intestinal metaplasia (n = 4), normal oesophagus (n = 5) or normal stomach (n = 1). DNA was extracted from three consecutive sections of each paraffin block and mutations at bases 1 and 2 of K-ras codon 12 were identified using a novel restriction endonuclease-mediated selective polymerase chain reaction method. RESULTS: Mutations were found in 7 of 23 (30.4%) adenocarcinomas and in 2 of 5 (40%) high-grade dysplasia specimens. No mutations were found in specimens of low-grade dysplasia, intestinal metaplasia without dysplasia, or normal oesophagus and stomach. There were no significant associations between the presence of mutations and clinicopathologic features in the patients with cancer. One patient who progressed from low-grade to high-grade dysplasia was found to have developed mutant K-ras in the course of this transformation. CONCLUSION: These results suggest that K-ras codon 12 mutations may occur frequently in patients with Barrett's oesophagus with high-grade dysplasia or adenocarcinoma of the oesophagus or oesophagogastric junction. K-ras mutation may be a late event in the Barrett's metaplasia-dysplasia-adenocarcinoma sequence.

Detection of rare mutant alleles by restriction endonuclease-mediated selective-PCR: assay design and optimization.

BACKGROUND: Restriction endonuclease-mediated selective (REMS)-PCR, allows detection of point mutations, deletions, and insertions. Reactions require concurrent activity of a restriction endonuclease (RE) and a DNA polymerase, both of which must be sufficiently thermostable to retain activity during thermocycling. The inclusion of the RE in REMS-PCR inhibits amplification of sequences containing the RE recognition site, thus producing selective amplification of sequences that lack the RE site. METHODS: Assays were used that allowed the selection of conditions that produce concurrent RE/DNA polymerase activity. The RE thermostability assay involved thermocycling a RE under various conditions and assessing residual cleavage activity at various time points. Conditions found to preserve RE activity during thermocyling were then tested for their compatibility with DNA polymerase-mediated PCR. RESULTS: A range of conditions that preserve activity of the RE BstNI over 30 cycles of PCR was identified. A subset of these conditions was subsequently found to mediate specific amplification using Taq DNA polymerase. These conditions were used to develop a REMS-PCR protocol for the detection of mutations at codon 12 of the K-ras gene. This protocol allowed the detection of 1 mutant allele in a background of 1000 wild-type alleles. The presence of primer sets for RE and PCR control amplicons provided unambiguous assessment of mutant status. CONCLUSION: Implementation of the assays described may facilitate development of REMS-PCR assays targeted to other loci associated with disease.

Object-based selection operates on a grouped array of locations.

In two experiments, space- and object-based selection effects were investigated, using variants of the ring-cuing paradigm of Egly and Homa (1984). The results revealed significant cuing modulation for nonring configurations of target locations spanning a range of retinal eccentricities, with the cuing effects independent of eccentricity and confined to the configuration of locations (rather than extending to locations within the space enclosed by the cued configuration). These results are consistent with object-based selection operating on a grouped spatial array (Vecera & Farah, 1994). Object selection may be based on a supradimensional saliency map representation of the field, modulated by feature-specific segmentation mechanisms (e.g., an object may be made salient on the basis of its color). Complex objects may be represented by grouped saliency signals. In this way, a two-dimensional spatial (saliency) representation may provide the common format for object-based selection, prior to full object definition.

Absolute quantitation of specific mRNAs in cell and tissue samples by comparative PCR.

A comparative PCR assay, for the absolute quantitation of specific mRNAs in cell and tissue samples, has been designed to overcome problems with previous techniques. cDNAs made from the RNAs are co-amplified with "competitor" plasmid templates under conditions in which reagents are not limiting at the equivalence point, thereby preventing competition between target and competitor templates and distinguishing the assay from competitive PCR assays. The cDNAs are serially diluted, and competitor templates concentrations are kept constant, rather than vice versa, as occurs in competitive PCR assays. Products from target and competitor templates are resolved by electrophoresis and measured by phosphorescent or fluorescent imagery. Both products are measured to minimize errors in the competitor:target ratio. A synthetic external standard RNA is included in the tissue lysis solution and co-purified with endogenous mRNAs, thereby being subjected to identical losses of yield during subsequent procedures. The determination of the number of copies of external standard cDNA allows inefficiencies of RNA extraction and cDNA synthesis to be taken into account. Standard concentrations of plasmids containing the endogenous target sequences are also measured, so that corrections can be made for discrepancies due to unequal amplification of target and competitor sequences. These corrections, together with the use of an external standard and the PCR conditions chosen, allow for the accurate, specific and sensitive determination of the absolute number of mRNA copies in a sample.

Pigmented adenoma mimicking a juxtapapillary melanoma. A 20-year follow-up.

A 52-year-old white woman was first diagnosed with a tumor of the right optic nerve in 1972. She remained asymptomatic until 1992, when she had a seizure on the left side of her body from a frontoparietal glioblastoma multiforme. Ophthalmic examination revealed enlargement of the eye tumor. This case provides clinical documentation spanning 20 years of a growing, pigmented tumor of the optic nerve head shown histopathologically to be a retinal pigment epithelial adenoma.

Epithelial cells up-regulate matrix metalloproteinases in cells within the same mammary carcinoma that have undergone an epithelial-mesenchymal transition.

A metastatic rat mammary carcinoma cell line, BC1, contains cells that have retained epithelial differentiation characteristics and metaplastic cells that have undergone an epithelial-mesenchymal transition. These two subpopulations cooperate to degrade collagen. We have used novel PCR assays to quantitate, for the first time, absolute levels of the mRNAs encoding matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) in cell and tumor samples. BC1 tumors expressed high levels of the collagenase-3, TIMP-2, stromelysin-1, and gelatinase B genes and low levels of the stromelysin-2 and TIMP-1 genes. This pattern of expression was repeated in cultures of BC1 and cultures containing mixed clones of epithelial cells and metaplastic cells. In both BC1 and the biclonal cultures, metaplastic cells were the main source of collagenase-3, stromelysin-1 and stromelysin-2, whereas TIMPs were equally distributed and epithelial cells were the main source of gelatinase B. High levels of all four MMP mRNAs in metaplastic cells were dependent on coculture with epithelial cells, suggesting the production of an inducing factor by the epithelial cells. In contrast, gelatinase B mRNA was produced at a high level by epithelial cells in the absence of metaplastic cells. TIMP-2 mRNA was abundant in both subpopulations grown alone and did not change substantially upon coculture. Thus, the interclonal cooperativity to degrade collagen in BC1 cells required the induction of MMPs in metaplastic cells by epithelial cells. Interclonal cooperativity may be important to the progression of neoplastic tumors, a feature of which is phenotypic heterogeneity.

Restriction endonuclease-mediated selective polymerase chain reaction: a novel assay for the detection of K-ras mutations in clinical samples.

The enriched polymerase chain reaction (PCR) assay has been used extensively in the detection of ras gene mutations in many types of human malignancies. Although it is very sensitive, it has a number of features that limit its use in the routine diagnostic laboratory. The aim of this study was to develop a novel enriched PCR strategy, in which the concurrent activity of the restriction enzyme BstNI and Taq polymerase allowed the amplification of mutant K-ras while inhibiting the formation of wild-type product. This restriction endonuclease-mediated selective PCR assay uses three sets of primers, together with BstNI, in the reaction mix, and the amplification products are analyzed by gel electrophoresis. The reliability of the restriction endonuclease-mediated selective PCR assay to detect activated K-ras was determined in a variety of clinical samples, including 139 fresh colorectal carcinomas and 113 paraffin-embedded blocks from 80 separate tumors of the colon and rectum, pancreas, breast, or kidney. Codon 12 mutations of the K-ras oncogene were identified in DNA from both fresh and paraffin-embedded tumors in a rapid, sensitive, and reproducible manner. Mutations were detected in 33 (24%) of the fresh colorectal cancers and 16 (20%) of the paraffin-embedded tumors. These results were 97% concordant in cases in which paraffin blocks and fresh specimens from the same tumor were available for analysis. We conclude that restriction endonuclease-mediated selective PCR is a sensitive, rapid, and robust assay for the detection of point mutations in a variety of clinical samples. Importantly, there is no need for manipulation of the sample once the PCR has been set up, and therefore, the chance of contamination is significantly reduced. In contrast to previous assays, restriction endonuclease-mediated selective PCR is not labor intensive, and its format is suitable for use in routine diagnostic laboratory.

Intrastromal clefts in keratoconus patients with hydrops.

PURPOSE: To report the clinical appearance and course of intrastromal clefts occurring with acute hydrops in keratoconus. METHODS: In eight patients with bilateral keratoconus, nine eyes developed acute corneal hydrops complicated by intrastromal cleft formation. One patient developed this complication in both eyes. The patients, three female and five male, had a mean age of 20 years (range, 11 to 36 years) and were followed after onset of acute hydrops for a median of 8 months (range, 2.5 to 21 months). The patients in this retrospective study were identified and had been treated at one of two institutional cornea referral practices. RESULTS: In eight of nine eyes with intrastromal clefts, complete cleft closure occurred between 6 weeks and 6 months. In one eye, cleft closure was nearly complete in a patient followed up for only 4.5 months after onset of hydrops. Corneal stromal neovascularization developed in six of the nine of eyes. At the last follow-up visit, four of the six untreated eyes had a best-corrected visual acuity of 20/200 or worse. The patient with intrastromal clefts in both eyes did not develop stromal neovascularization and achieved a contact lens corrected visual acuity of 20/40 or better in each eye without surgical intervention. CONCLUSIONS: Intrastromal cleft formation is a manifestation of corneal hydrops in keratoconus. Single or multiple clefts can occur, and bilateral involvement is possible. Clefts generally close over a period of months, but stromal neovascularization is common and may compromise future graft survival.

Methicillin-resistant Staphylococcus aureus in a high school wrestling team and the surrounding community.

OBJECTIVES: To describe a community outbreak of methicillin-resistant Staphylococcus aureus (MRSA) and to investigate risk factors for MRSA transmission and infection in a wrestling team. DESIGN: Case series and retrospective cohort study. SETTING: A high school wrestling team and the surrounding community in southern Vermont, 1993 to 1994. PATIENTS OR OTHER PARTICIPANTS: The case series included persons whose MRSA-positive infections were identified at a hospital laboratory from January 1, 1993, through February 28, 1994, and a health maintenance organization laboratory from July 1, 1993, through February 28, 1994. A wrestling team case-patient was a 1993-1994 team member with an MRSA-positive culture during the period from January 1, 1993, through February 28, 1994. INTERVENTIONS: Visual inspection of wrestlers before matches was instituted. Affected wrestlers were excluded from wrestling and advised to seek appropriate medical care. Heightened attention was given to personal and environmental hygiene. MAIN OUTCOME MEASURES: Colonization or infection with MRSA. RESULTS: Seven of 32 team members were MRSA positive (6 infected, 1 colonized). All lesion-positive wrestlers were tested by pulsed field gel electrophoresis and found to be infected with the same MRSA strain, as were 6 nonwrestlers. No risk factors for MRSA infection were identified. CONCLUSIONS: The MRSA was transmitted among members of a wrestling team. Infection with MRSA should be suspected in outbreaks of boils that are nonresponsive to standard antibiotic therapy among healthy participants of contact sports and their close contacts.

Serum-free culture of mouse tracheal epithelial cells.

Numerous investigators have described maintenance of airway epithelial cells from various species in a differentiated state in primary culture. Because the number of cells that can be isolated from the mouse trachea is very small, published techniques are unsuitable for this species. To examine the production of growth factors by murine airway epithelial cells, the authors developed a method for culture of mouse tracheal epithelial cells from explants, in which the population of cells was expanded in the presence of epidermal growth factor and insulin-like growth factor-I, which exhibited synergistic mitogenic activity. After subculture, an essentially pure population of epithelial cells was recovered, with a yield approximately tenfold greater than reported using protease dissociation of cells from the trachea. Culture of the cells at passage 2 on a collagen gel substratum induced differentiation toward a synthetic/secretory phenotype, accompanied by marked diminution in spontaneous and mitogen-induced DNA synthesis without loss of viability. In parallel, secretion of immunoreactive transforming growth factor-beta by the epithelial cells was strikingly increased, but could be partially down-regulated in the presence of mitogenic growth factors.

Epithelial cell-derived transforming growth factor-beta in bleomycin-induced pulmonary injury.

We have investigated whether enhanced secretion of transforming growth factor-beta (TGF-beta) by distal respiratory epithelial cells was associated with the development of bleomycin-induced pulmonary fibrosis. Type 2 pneumocyte-enriched preparations of bronchioloalveolar epithelial cells from normal mouse lung tissue released latent TGF-beta when cultured in serum-free medium. TGF-beta in culture supernatants could be detected using a sensitive enzyme immunoassay which employed enzyme complex amplification as a reporter system, as well as by a radiolabelled receptor competition assay. Exposure to bleomycin and other potentially fibrogenic stimuli in vitro did not stimulate production of TGF-beta by the epithelial cells but release was enhanced by treatment of the cells with interferon-gamma. Type 2 pneumocyte-enriched cell preparations obtained following induction of a pulmonary inflammatory response by administration of intratracheal bleomycin to susceptible C57BL/6 mice did not demonstrate increased release of TGF-beta in culture. However, the concentration of TGF-beta in bronchoalveolar lavage (BAL) fluids was significantly elevated compared to controls at 1 and 2 weeks after bleomycin-induced injury in these mice. No such increase was detected in BAL fluids from BALB/c mice, which are resistant to the effects of bleomycin. These results provide no support for a pathogenetic role of alveolar epithelial cell-derived TGF-beta in bleomycin-induced pulmonary fibrosis. Nevertheless, elevated levels of TGF-beta in BAL fluids may provide a marker of the progression of pulmonary injury to fibrosis.

Epidermal growth factor-like molecular species in normal bronchoalveolar lavage fluid.

Normal bronchoalveolar lavage fluid (BALF) contains mitogenic activity for fibroblasts and type 2 pneumocytes. A number of growth factors that might contribute to this activity have been identified in BALF. We found that a molecule or molecules able to bind to epidermal growth factor (EGF) receptors on mouse lung fibroblasts were present in normal mouse BALF and could be blocked by an antiserum to mouse EGF. Receptor binding was partially blocked by preincubation with heparin, indicating a relationship to the heparin-binding subgroup of EGF-like growth factors. Heparin markedly enhanced the mitogenic activity of BALF for fibroblasts, but we were unable to establish whether the EGF-like molecule contributed to this activity. Immunoblotting using the anti-EGF serum identified a protein of M(r) 88,000 in concentrated BALF. The cellular source and physiological role of this growth factor merit further investigation.

The prediction of long-term functional outcomes of children with cerebral palsy.

Current information was obtained from 117 respondents, including 71 who completed interviews, of a population of 810 individuals who had been diagnosed with cerebral palsy between 1951 and 1974. Original chart reviews constituted the basis for predicting employment capabilities of the interviewed cohort. A comparison of predictions with actual functional outcomes demonstrated a tendency to pessimistic underestimation of long-term functional outcome, sounding a note of caution to paediatric developmentalists.

Cat model for intraocular pressure reduction after transscleral Nd:YAG cyclophotocoagulation.

Transscleral neodymium:yttrium-aluminum-garnet (Nd:YAG) laser cyclophotocoagulation (TSNYC) is used to lower intraocular pressure (IOP) in glaucoma patients refractory to conventional medical and surgical therapy. Our study investigates the ability of TSNYC to lower IOP in normal cats. One eye of 13 cats was treated with non-contact TSNYC. Mean pretreatment IOP was 25 mm Hg (vs. 25.3 mm Hg in contralateral control eyes). Eyes received 80 laser applications over 360 degrees delivered at least 3 mm posterior to the limbus with maximum power (8 to 9 joules) and maximum retrofocus (3.6 mm). Eyes were retreated if IOP was not reduced below baseline after 2 weeks. By 4 weeks, IOP was decreased in all treated eyes by a mean of 29.2% and was maintained as long as 20 weeks (mean decrease 14.8%). IOP in 10 eyes was lowered after a single treatment session. Of these, 2 eyes had IOP spikes > 10 mm Hg prior to IOP reduction. Three cats required retreatment to maintain IOP reduction. All eyes developed transient (< 4 weeks) postoperative uveitis and 3 eyes developed rubeosis iridis which resolved with topical corticosteroids. Histologic examination (6 weeks post-treatment) showed focal disruption of the pigment epithelium and to a lesser degree the nonpigmented epithelium at the base of the ciliary body and in the valleys of the pars ciliaris. The epithelium at the apices of the ciliary processes appeared intact. Vascular engorgement was variably present. This study demonstrates that TSNYC lowers IOP in cats. This animal model will be useful for investigating mechanism(s) responsible for TSNYC-induced IOP reduction.

Maintenance of differentiated phenotype by mouse type 2 pneumocytes in serum-free primary culture.

An improved method has been developed for separation of an enriched population of mouse type 2 pneumocytes, based on differential adherence and size fractionation of cells dissociated with trypsin. These cells were successfully maintained in primary culture in serum-free medium MCDB 201 supplemented with albumin, transferrin, and lipids. Whereas type 2 pneumocytes in serum-supplemented culture undergo phenotypic transformation into adherent flattened cells that resemble type 1 pneumocytes, this did not occur in serum-free culture. Both the morphology of the type 2 pneumocytes and their expression of surfactant protein A were maintained for at least 6 days in vitro. However, rapid loss of differentiated characteristics was induced by exposure of the cells to normal mouse serum. This was accompanied by a striking decrease in spontaneous DNA synthesis as assessed by incorporation of tritiated thymidine. When cultured in serum-free medium, the behavior of the type 2 pneumocytes on various extracellular matrix components was different from that reported for serum-supplemented culture. Serum-free culture of type 2 pneumocytes may offer significant advantages for evaluation of the secretory activities of these cells in vitro.

Collaboration in investigator initiated public health nursing research: university and agency considerations.

There is a significant difference in the collaborative process between whether a public health nursing research project is requested (of the researchers) by the agency or if it is conceived by an outside investigator. This article discusses the underlying concepts of negotiation, mutuality, and respect that support the process of an externally initiated study in an agency. In a progressive listing format, the important components within the planning, development, implementation, and completion phases are then described so that they can be useful to beginning researchers as a guide and to experienced researchers as a reminder.

Immunoenzymatic detection of collagen production by pulmonary fibroblasts in serum-free culture.

We have developed an improved assay for the production of collagen by fibroblasts. Early passage adult mouse lung fibroblasts, established and maintained in serum-free culture, were employed as the target cells. An enzyme immunoassay was used for detection of type I collagen deposited on the substratum, permitting adaptation of the technique to cultures in 96-well microplates. Approximately two-fold enhancement of collagen deposition was induced by exposure to a concentration of 3 ng/ml of transforming growth factor-beta 1 or of 100 ng/ml of insulin-like growth factor-1 for 48 hr.