Hyperprolactinemia and hypothyroidism following cytotoxic therapy for central nervous system malignancies.

Endocrinologic dysfunction including hyperprolactinemia and hypothyroidism are recognized complications of irradiation to the hypothalamic-pituitary axis or thyroid gland in the course of treating CNS malignancies. However, the frequency of these adverse effects in both short- and long-term survivors may be underestimated. Sixty-five patients treated in the University of Rochester Cancer Center since 1968 with radiation with or without BCNU chemotherapy for CNS tumors not involving the hypothalamic-pituitary axis were evaluated for thyroid, prolactin, and gonadal disturbances regardless of clinical symptomatology. Prolactin values were elevated in 19 of 47 patients (40%). For males and females treated with greater than 55 Gy, abnormal values were present in nine of 11 (82%) and seven of 14 (50%), respectively. For males and females treated with less than or equal to 55 Gy, two of nine (22%) and one of 13 (8%), respectively, were abnormal (P = .0001). Six of six patients who also received BCNU chemotherapy were hyperprolactinemic, as compared with six of ten (60%) who did not receive BCNU. Seven of eight females with elevated prolactin levels had menstruation abnormalities, and five of seven adult males noted a decrease in libido. Mild abnormalities in testosterone concentration were found in three of nine men evaluated, all of whom had normal gonadotropins. Of 47 patients who did not receive irradiation to the spinal axis (and thus the thyroid gland), ten (21%) had a decreased thyroxin (T4) value. Only one of these patients had an elevated thyroid-stimulating hormone (TSH) value. Of 32 patients who received greater than 55 Gy, ten (31%) had a low T4, compared with zero of 15 who received less than or equal to 55 Gy (P = .0001). Four of eight patients (50%) who also received BCNU had low T4 values, as compared with three of 14 (21%) who did not receive BCNU. Of 15 patients who were treated with 4 to 10 MV photon irradiation to the spinal axis, five patients (33%) had elevated TSH values. The mean spinal axis dose in these patients was 33 Gy. Two euthyroid children in this group manifested the early onset of puberty. The complex of endocrinologic abnormalities observed in several patients receiving only cranial irradiation, that is elevated prolactin, decreased thyroid, and gonadal hormone secretion in the presence of otherwise normal pituitary hormone levels, suggests a radiation-induced insult to the hypothalamic regulation of pituitary function.

Real-time microbe detection based on director distortions around growing immune complexes in lyotropic chromonic liquid crystals.

We describe director distortions in the nematic liquid crystal (LC) caused by a spherical particle with tangential surface orientation of the director and show that light transmittance through the distorted region is a steep function of the particle's size. The effect allows us to propose a real-time microbial sensor based on a nontoxic lyotropic chromonic LC (LCLC) that detects and amplifies the presence of immune complexes. A cassette is filled with LCLC, antibody, and antigen-bearing particles. Small and isolated particles cause no macroscopic distortions of the LCLC. Upon antibody-antigen binding, the growing immune complexes distort the director and cause detectable optical transmittance between crossed polarizers.

Absence of prognostic significance, peritoneal dissemination and treatment advantage in endometrial cancer patients with positive peritoneal cytology.

Peritoneal cytology has been shown to be one of the prognostic factors in endometrial cancer. A series of 134 patients was seen between January 1977 and March 1985 with clinical Stage I (or treated as a clinical Stage I) endometrial adenocarcinoma at the University of Rochester Cancer Center. The majority of patients underwent extrafascial hysterectomy with the majority of washings obtained at the time of surgery. Fourteen percent (19/134) of the patients were found to have positive cytology. Eleven patients with positive cytology (11/19) were treated with local-regional pelvic treatment; the other eight patients received whole abdominal therapy. The recurrence rates were less with the local treatment than with the whole abdominal treatment groups (9.1% vs. 25%) in those patients having positive cytology. There was no statistical difference in recurrence rates between the pathologic Stage I patients with positive cytology (10%) versus those patients having negative cytology (5%), nor was there statistical difference in survival between pathologic Stage I positive or negative cytology patients. It is suggestive from this non-randomized study that positive cytology in endometrial cancer is not an independent prognostic factor and that whole abdominal irradiation did not influence outcome.

Treatment of breast cancer among elderly women with segmental mastectomy or segmental mastectomy plus postoperative radiotherapy.

Elderly women with cancer are often treated non-aggressively. Between January 1972 and March 1984, 128 women greater than 60 years were treated for Stage I or II breast cancer with segmental mastectomy (SGM) plus/minus postoperative radiation at one of our four area hospitals. Whereas 82% of similar patients less than 60 years were referred for postop radiation, only 39.8% of patients greater than 60 were so referred. Referral rates progressively diminished with increasing patient age above 60. Thus, we reviewed the outcome of 77 elderly women treated with SGM and 51 treated with SGM+RT. Treatment groups were similar with regard to prognostic clinical and histologic parameters. Mean follow-up is 51.4 months. Among SGM patients, 45.5% of patients between 60-70 years, 37.9% of those greater than 70, and 20% of those greater than 80 years experienced loco/regional failure prior to death. Conversely, only two local failures occurred among all elderly women treated with SGM+RT. Distant failure was approximately 11% and was unaffected by treatment. Complications of SGM+RT were modest. These data suggest that SGM+RT can be safely and effectively applied to the elderly. Moreover, the data suggests that postop radiation may be more beneficial when extended to elderly patients post segmental mastectomy than among younger women. Referring surgeons should focus upon their patients' physiologic and not chronologic age as a basis for treatment allocation decisions.

Improved immunoelectron microscopic method for localizing cytoskeletal proteins in Lowicryl K4M embedded tissues.

We have modified the Lowicryl K4M low-temperature dehydration and embedding procedure for immunoelectron microscopy to provide improved ultrastructural detail and facilitate the localization of actin and tubulin in isolated rat adrenocortical cells, chick spinal cord with attached dorsal root ganglia (SC-DRG), and cultured dorsal root ganglia (DRG). Cells and tissues were fixed for immunocytochemistry either in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde (0.1 M PIPES buffer, pH 7.3) or in a mixture of 0.3% glutaraldehyde and 1.0% ethyldimethylaminopropylcarbodiimide (0.1 M phosphate buffered saline, pH 7.3). Dehydration was in ethanol at progressively lower temperatures to -35 degrees C. Infiltration at -35 degrees C was followed by ultraviolet polymerization at -20 degrees C. Comparable samples were fixed in glutaraldehyde and osmium tetroxide and embedded in Epon 812 or Epon-Araldite. Post-embedding immunostaining of thin sections utilized commercially available monoclonal antibodies to tubulin and actin followed by the protein A-gold technique (Roth et al., Endocrinology 108:247, 1981). Actin immunoreactivity was observed at the periphery of mitochondria and between mitochondria and lipid droplets in rat adrenocortical cells and at the periphery of neuronal cell processes of SC-DRG. Tubulin immunoreactivity was associated with microtubules throughout neurites of cultured DRG. Our modified technique allows preservation of ultrastructural details as well as localization of antigens by immunoelectron microscopy.

Differences in integrin expression during avian corneal stromal development.

PURPOSE: The purpose of this study was to determine whether there are changes in integrin expression associated with the spatial and temporal variations in matrix expression that occur during specific stages in corneal stromal development. METHODS: Immunofluorescence techniques were used to analyze beta 1-containing integrins and alpha v beta 3 localization both in situ and in cell cultures. RESULTS: In situ, beta 1 and alpha v beta 3 were present with different patterns of localization, and these varied with developmental stage. beta 1-containing integrins were present on most cells, whereas alpha v beta 3 was present on cells at the corneal-scleral epitheliomesenchymal interface during migration of keratocyte precursors; very little alpha v beta 3 was localized in keratocytes. Keratocytes and undifferentiated periocular mesenchyme cells grown in vitro also exhibited differences in localization of beta 1-containing integrins and alpha v beta 3. All focal adhesions contained beta 1, whereas a subset contained both beta 1 and alpha v beta 3, indicating potential functional differences in focal adhesions. In addition, most periocular mesenchyme cells exhibited alpha v beta 3-containing focal adhesions throughout, but the majority of keratocytes contained only peripherally located alpha v beta 3-positive focal adhesions. The localization of both beta 1-containing integrins and alpha v beta 3 was modulated by time allowed for attachment and spreading. CONCLUSIONS: Keratocytes and undifferentiated periocular mesenchyme cells exhibit developmental differences in integrin localization in situ. These two cell types also exhibit different patterns of alpha v beta 3 localization in vitro, possibly as a result of developmental differences in ligand-binding properties. beta 1-containing integrins and alpha v beta 3 define different types of focal adhesions, implying different functions. These differences in expression may be important in the initiation of cellular migration in the early stages of corneal development, as well as in the transition from the undifferentiated to the differentiated keratocyte phenotype.

Identification and functional characterization of two type VI collagen receptors, alpha 3 beta 1 integrin and NG2, during avian corneal stromal development.

PURPOSE: The development and maintenance of extracellular matrix architecture in the corneal stroma is associated with abundant type VI collagen deposition. This collagen has been implicated in mediating both cell-matrix and matrix-matrix interactions. Although corneal fibroblasts spread extensively on this collagen, its role in corneal development has not been elucidated. METHODS: To clarify the role of this collagen, two type VI collagen receptors were studied during corneal development using immunochemical techniques: alpha 3 beta 1 integrin and an integral membrane proteoglycan, NG2. RESULTS: At embryonic day 6, these receptors were present in a diffuse pattern on cells within the cornea and juxtacorneal regions, indicating a migratory phenotype. At embryonic day 14, when the stroma is fully differentiated, alpha 3 and NG2 were localized in a punctate pattern on a subset of corneal fibroblasts, whereas beta 1 was more ubiquitously expressed. Colocalization of NG2 and type VI collagen indicated that this collagen was present and punctate in its organization was associated with NG2-positive cells. Immunochemical analyses at embryonic days 5 and 14 revealed alpha 3 and beta 1 at 155 kDa and 120 kDa, respectively, and demonstrated that these subunits were interacting to form a heterodimer. NG2 was present with a core protein of 330 kDa and an intact proteoglycan of approximately 600 kDa, and analysis of stromal lysates indicated a chondroitin sulfate-containing proteoglycan. Matrix-receptor cross-linking demonstrated the interaction of beta 1 and NG2 in periocular mesenchyme cells and corneal fibroblasts with type VI collagen, whereas only a subset of cells expressed alpha 3, indicating the presence of another beta 1 integrin. No variations between in vivo and in vitro expression of either alpha 3 beta 1 or NG2 were observed. CONCLUSIONS: These data indicate that two receptors for type VI collagen, alpha 3 beta 1 and NG2, are present during corneal stromal development, with a functional interaction of these receptors with type VI collagen. These interactions may play a role in corneal cell migration, development, and maintenance of corneal architecture.

Treatment of breast cancer with segmental mastectomy alone or segmental mastectomy plus radiation.

A retrospective review of the outcome of treatment for primary, Stage I and II breast cancer with segmental mastectomy (SGM) alone or segmental mastectomy plus postoperative irradiation (SGM + RT) at four Rochester, New York, city hospitals is reported. Between January 1971 and March 1984, 99 women were treated with SGM and 146 with SGM + RT. Groups were similar regarding significant clinical and histologic prognostic factors; they differed, however, in that the SGM group was considerably older (means = 72) than the SGM + RT group (means = 56). Among SGM patients, local and total locoregional failure was 26.44 and 35.2%, respectively. Local and total locoregional failure (7.6 and 12.4%, respectively) was significantly reduced among patients treated with SGM + RT (p less than 0.0001). Among SGM patients, there was scant advantage in enlarging the extent of resection from local excision (29.5% local failure) to wide local excision (27.3%) to quadrantectomy (22.2%). Among women receiving SGM + RT, similar rates of local failure occurred among patients receiving local excision (15.5%) and wide local excision (12.5%). By contrast, only 2.8% of those receiving quadrantectomy failed. Results are viewed as supportive of findings of NSABP-B06. Findings suggest that SGM constitutes inadequate treatment of Stage I and II breast cancer. Locoregional failure rates of 30-40% may be reduced to around 10% with postoperative irradiation.

The effects of nerve growth factor and dibutyryl cyclic AMP on cytoskeletal densities in cultured sensory ganglia.

The effects of nerve growth factor (NGF) and dibutyryl cyclic AMP (DBC) on the density of cytoskeletal structures in cultured dorsal root ganglia were examined using morphometric techniques. After 24 hr in culture, NGF-treated neurites were longer than either DBC-treated or control neurites. At 48 hr, neurites produced in response to NGF and DBC were of equivalent length, while controls were considerably shorter. Comparison of electron micrographs of neuritic profiles revealed some differences of area and cytoskeletal density between treatment groups. Morphometric analysis was used to determine these differences under several growth conditions, at various rates of elongation and at different neurite lengths. As shown by analysis of variance, both NGF-treated and control neurites tapered in diameter at 48 hr in vitro, while DBC-induced neurites increased in area. An increase in cytoskeletal density for all treatment groups indicated that density was not always correlated with changes in area. An increased density of microtubules as compared to neurofilaments was seen at 24 hr, with equal densities of both cytoskeletal elements present after 48 hr in vitro. Comparisons between individual groups of data indicated that NGF-treated neurites relied primarily on microtubular density at 24 hr in vitro, when NGF induced longer, faster growing neurites. At 48 hr, there was an increase in neurofilaments proximal to the explant in the presence of DBC, implying that DBC may cause increased synthesis and/or transport of these structures. A comparison of microtubule to neurofilament ratios indicated that at 24 hr, there was always a greater density of microtubules. However, after 48 hr, neurofilament density increased such that there were equivalent densities of both cytoskeletal elements, possibly due to the overall increase in length observed in each treatment group. These data imply that 1) neurites with different rates of elongation may exhibit differences in cytoskeletal density; 2) neurites of equivalent lengths may be of differing stabilities; 3) NGF and DBC produce neurites with different cytoskeletal densities, implying divergent mechanisms of neurite induction; 4) the presence or absence of NGF may be partially responsible for variations in cytoskeletal densities observed between peripheral and central processes of DRG during development.

The impact of OBRA-87 on psychotropic drug prescribing in skilled nursing facilities.

OBJECTIVES: This study examined the impact of regulations established by the Omnibus Budget Reconciliation Act of 1987 (OBRA-87) on prescriptions for psychotropic drugs, and on research on their use in nursing homes. METHODS: Data were collected on drugs prescribed for residents of 39 skilled nursing facilities over the four-year period from 1989 to 1992, bracketing the implementation of OBRA-87 in the fall of 1990. Changes in prescribing patterns were analyzed by drug class, specific target medications and doses, number of drugs prescribed, and multidrug combinations. To determine the effect of OBRA-87 on research, peer-reviewed journals were searched for the number and content of publications on psychotropic drug use in skilled nursing facilities between 1980 and 1996. RESULTS: The number of prescriptions for antipsychotics, sedative antihistamines, and sedative-hypnotics decreased significantly, while prescribing of anxiolytics increased. Qualitative, but not quantitative, shifts occurred in prescriptions for antidepressant drugs, the most frequently used psychotropic medications in all years. Rates of psychotropic polypharmacy remained stable. The number of data-based publications on psychotropic drug use in nursing homes increased after implementation of OBRA-87, but few were related to the effectiveness of drug treatment. CONCLUSIONS: Implementation of OBRA-87's nursing home regulations was associated with reductions in use of drugs specifically targeted by this legislation and was a potent stimulus to research, an unanticipated benefit of legislative action. Increased use of anxiolytics, persistent prescribing of anticholinergic antidepressants, enthusiastic adoption of new agents despite a limited research database involving frail patients, and the paucity of new studies reporting data on clinical effectiveness suggest a need for targeted research on treatment outcomes to improve the care of this population.

Fibroblasts retain their tissue phenotype when grown in three-dimensional collagen gels.

Fibroblasts are responsible for the synthesis, assembly, deposition, and organization of extracellular matrix molecules, and thus determine the morphology of connective tissues. Deposition of matrix molecules occurs in extracellular compartments, where the sequential stages are under cellular control. Cell orientation/polarity is important in determining how the cell orients these extracytoplasmic compartments and therefore how the matrix is assembled and oriented. However, the control of cell orientation is not understood. Fibroblasts from three tissues with different morphologies were studied to determine whether cells maintained their characteristic phenotype. Fibroblasts from cornea, which in vivo are oriented in orthogonal layers along with their matrix; from tendon, a uniaxial connective tissue, where cells orient parallel to each other; and from dermis, a connective tissue with no apparent cellular orientation, were used to study cell morphology and orientation in three-dimensional collagen gels. The different cells were grown for 3 and 7 days in identical three-dimensional collagen gels with a nonoriented matrix. Confocal fluorescence microscopy demonstrated that corneal fibroblasts oriented perpendicular to one another at 3 days, and after 7 days in hydrated gels these cells formed orthogonal sheets. Tendon fibroblasts were shown by the same methods to orient parallel to one another in bundles at both 3 and 7 days, throughout the depth of the gel. Dermal fibroblasts showed no apparent orientation throughout the hydrated gels at either time point examined. The organization of these different cell types was consistent with their tissue of origin as was the cell structure and polarity. These studies imply that cellular and tissue phenotype is innate to differentiated fibroblasts and that these cells will orient in a tissue-specific manner regardless of the extracellular matrix present.

Type VI collagen increases cell survival and prevents anti-beta 1 integrin-mediated apoptosis.

Cell-matrix interactions are important in the development of the avian cornea. Type VI collagen is present within the periocular mesenchyme prior to the migration of cells into the corneal stroma and is abundant in the mature stroma. Whether the interaction of cells with type VI collagen is essential for cellular survival in the cornea is not known. In the present study, we examined the interaction of corneal cells with type VI collagen in vitro to determine if it can increase cell proliferation and decrease apoptosis. In vivo analysis demonstrated that apoptosis occurs in the periocular region during early stages of avian corneal development, but in fully mature corneas apoptosis only occurs in the corneal epithelium and not in the stroma. In vitro analysis examined the importance of beta 1 integrin interactions with type VI collagen in mature corneal fibroblasts and the precursor cells. Using an anti-beta 1 integrin blocking antibody, CSAT, integrin/matrix interactions were disrupted. Results indicated that viability of both corneal fibroblasts and periocular mesenchyme cells was greater on type VI collagen than on type I collagen or BSA-blocked glass. In addition, less apoptosis was observed for both cell types on type VI collagen when beta 1 integrin--matrix interactions were disrupted. These data indicated that these cells require intact beta 1 interactions with type I collagen and with BSA-coated glass controls to remain viable. Thus, type VI collagen may play a role in the rescue of corneal cells from anti-beta 1 integrin-induced apoptosis by increasing cell survival, probably via a non-beta 1 integrin-dependent mechanism.

Antidepressant prescribing in nursing homes: is there a place for tricyclics?

OBJECTIVE: To deduce a model describing physicians' choice of antidepressants for treating elderly nursing home patients. METHODS: Subjects were geriatric residents of 137 skilled nursing facilities who regularly received an antidepressant medication for at least one month (n = 3,440, 28% of all residents). Reasons for prescribing antidepressants and physicians' diagnoses of depression and dementia were identified by medical record audit. Residents were grouped by dementia and antidepressant target symptoms (depression, or one or more non-psychiatric symptoms, i.e. insomnia, pain, incontinence, itching). RESULTS: Selective serotonin reuptake inhibitors (SSRIs) were prescribed preferentially over tricyclic antidepressants (TCAs) for treating depression in both demented and non-demented residents, but TCAs were nine times more likely to be prescribed for treating non-psychiatric target symptoms alone. When non-psychiatric target symptoms were present without depression or dementia, both amitriptyline and nortriptyline prescribing was increased, but amitriptyline appeared to be the antidepressant of choice. In all subgroups examined, its use was two to five times more prevalent when such symptoms were present. In patients with dementia, amitriptyline prescribing declined whether or not non-psychiatric target symptoms were present, but nortriptyline prescribing did not; nortriptyline was three times more likely than amitriptyline to be prescribed in the presence of dementia. CONCLUSIONS: Physicians prescribe anticholinergic TCAs principally to treat common non-depressive symptoms in nursing home residents, preferring SSRIs for uncomplicated depression and depression with dementia. They tend to avoid prescribing anticholinergic TCAs other than nortriptyline when they recognize a patient as demented. The data suggest that physicians employ a decision model for antidepressant prescribing that simultaneously recognizes the utility of TCAs in treating non-psychiatric symptoms and the anticholinergic vulnerability of older, especially demented, patients. Whether or not this model leads to optimal patient management requires further study.

Ultrastructural and cytochemical evaluation of sepsis-induced changes in the rat pulmonary intravascular mononuclear phagocytes.

Sepsis stimulates an increase in the number and activity of mononuclear phagocytes in systemic host-defence organs. The present study was conducted to define the ultrastructural and cytochemical characteristics of the mononuclear phagocytes that sequester in the lung microvasculature of septic rats. Fourteen rats were challenged with a single intraperitoneal injection of saline (0.5 ml/100 g), E. coli (2 x 10(7)/100 g) or glucan (4 mg/100 g), and euthanased 2, 4, or 7 d later. The lungs were inflation fixed and processed for transmission electron microscopy. Cellular morphology was used to identify the intravascular mononuclear phagocytes and acid phosphatase (AcPase) expression was monitored as an index of cellular differentiation and activation. Control rats contained a limited number of monocytes in the pulmonary vasculature. In contrast, large numbers of activated mononuclear phagocytes were seen in the microvasculature within 48 h of treatment with either microbial product. The recruited pulmonary intravascular mononuclear phagocytes (PIMP) exhibited AcPase-reactive Golgi complexes, accumulation of secretory vesicles and other features of cell activation consistent with enhanced biosynthetic activity. Subsequent electron microscopy, conducted 4 and 7 d posttreatment, suggested that a progressive decline in the number and activity of PIMPs then occurred. In order to quantify the sepsis-induced accumulation of AcPase-positive PIMP, the experimental challenges were repeated in 11 rats and, 48 h later, tissue samples were evaluated by light microscopy for tartrate-insensitive acid phosphatase. Control rats exhibited 0.148 +/- 0.107 AcPase-positive PIMP/alveoli. E. coli and glucan challenged animals exhibited significant (P < 0.01) increases in AcPase-positive mononuclear phagocytes, with 0.782 +/- 0.073 and 0.636 +/- 0.170 PIMP/alveoli respectively. The results demonstrate that focal sepsis stimulates a significant, but transient, recruitment of activated mononuclear phagocytes into the rat pulmonary microvasculature.

Expression of integrins during axolotl limb regeneration.

Limb regeneration in urodeles is achieved through the dedifferentiation of tissues at the amputation plane and through the production of the blastema. This tissue breakdown is possible by extensive alterations in molecules of the extracellular matrix. In this respect we describe the regulation of several integrins during such events. It was found that alpha 1 and beta 1 integrins were down-regulated as blastema formation proceeded. In contrast, the expression of alpha 3, alpha 6 and alpha v integrins were upregulated in the blastema. These data are consistent with the roles of integrins in developmental phenomena and are discussed in light of the mechanisms of dedifferentiation.

Corneal cell-matrix interactions: type VI collagen promotes adhesion and spreading of corneal fibroblasts.

Type VI collagen is a nonfibrillar collagen present as a network throughout the chick secondary stroma. Immunolocalization of type VI collagen both in the chick corneal stroma and in other systems demonstrates that type VI collagen is present associated with cells and between striated fibrils. We hypothesize that type VI collagen may function in cell-matrix interactions important in corneal development. To examine this possibility, we have isolated and characterized bovine corneal type VI collagen and determined that the chain composition and morphology of type VI collagen isolated from cornea is similar to that isolated from other sources. The tissue form of type VI collagen was localized to filaments forming a network around fibrils and close to corneal fibroblasts. We then analyzed relative attachment and spreading on type VI collagen as compared to the other collagens present in the secondary stroma, and found that although corneal fibroblasts attach equally well to type VI and type I collagen, cells spread to a much greater extent on type VI collagen. Although corneal fibroblasts do have an RGD-dependent receptor which functions during adhesion to fibronectin, attachment to type VI collagen is RGD-independent unless the molecule is denatured. Blocking of the RGD-dependent receptor with soluble RGD peptides results in no change in attachment or spreading. These data imply a role for type VI collagen in cell-matrix interactions during corneal stroma development.

Spatial and temporal variations in extracellular matrix of periocular and corneal regions during corneal stromal development.

The development of the avian corneal stroma occurs in discrete developmental stages. During this sequence of events, the neural crest-derived corneal fibroblast precursor cells are surrounded by distinct extracellular matrices which change both spatially and temporally. To elucidate the role of these matrices, extracellular matrix components in the periocular mesenchyme and cornea were analysed prior to and during migration and differentiation of corneal fibroblasts using antibodies against collagens, proteoglycans and glycoproteins. Previous work has concentrated on the matrix of the corneal stroma rather than the matrix of the periocular mesenchyme. Since the precursors of the corneal fibroblasts are present within the must migrate through the periocular mesenchyme prior to entry into the cornea proper, this environment was fully evaluated. The present study documents the matrix composition of both the cornea and periocular mesenchyme at developmental stages that are prior to and after initiation of corneal invasion by the corneal fibroblast precursors. Variations in matrix molecules comprising both the periocular mesenchyme and cornea were demonstrated. These include changes in the distribution of collagen types I, II, III, IV and VI; the proteoglycans decorin and lumican; as well as the adhesive glycoproteins tenascin, fibronectin and laminin. It is hypothesized that the variations in matrix localization are important in the regulation of cell migration and differentiation during normal corneal development. Any regulation is likely to involve a combination of components found in the extracellular matrices and therefore, a consideration of the matrix rather than isolated components is required.

Blood cholesterol concentration: fingerstick plasma vs venous serum sampling.

To assess whether venous and fingerstick blood samples yield similar cholesterol concentrations, we obtained both types of samples simultaneously in 108 volunteers participating in a cholesterol screening program. All samples were analyzed by the same enzymatic method in a standardized laboratory, and pairs of simultaneous samples were measured in the same laboratory run. Cholesterol concentrations in fingerstick-derived plasma were consistently higher than in the venous serum (P less than 0.0001), by a positive bias averaging 3.6%. Cholesterol values in fingerstick plasma also were higher than cholesterol results for venous serum placed in a capillary collection tube (average bias +2.4%). The positive bias of fingerstick plasma vs venous serum results appears to be at least partly due to specimen handling, although a true physiological difference between venous and fingerstick cholesterol concentrations is probably also involved. If a positive bias of this magnitude from fingerstick blood sampling is left unadjusted, substantial numbers of people will be labeled "at risk" and referred to physicians when their true values were actually within the acceptable range.

A method for exposing the internal anatomy of small and delicate tissues for correlated SEM/TEM studies using polyethylene glycol embedding.

A method for preparing and handling large, clean, distortion-free cut surfaces through small and delicate tissues for correlated SEM/TEM examination is described. In this method, tissues are fixed according to conventional protocols; however, instead of critical-point-drying after fixation, tissues are first embedded in polyethylene glycol (PEG), a water-soluble waxy solid. Tissue blocks are easily oriented and sectioned to the desired regions, immersed in a solvent to remove PEG, critical-point-dried, and examined with an SEM. The same tissue blocks can be reworked for TEM by immersing in propylene oxide and embedding in an epoxy resin.

Collagen fibrillogenesis in vitro: interaction of types I and V collagen regulates fibril diameter.

The small-diameter fibrils of the chick corneal stroma are heterotypic, composed of both collagen types I and V. This tissue has a high concentration of type V collagen relative to other type I-containing tissues with larger-diameter fibrils, suggesting that heterotypic interactions may have a regulatory role in the control of fibril diameter. The interactions of collagen types I and V were studied using an in vitro self-assembly system. Collagens were purified from lathyritic chick embryos in the presence of protease inhibitors. The type V collagen preparations contained higher molecular weight forms of the alpha 1(V) and alpha 2(V) chains constituting 60-70% of the total. Rotary-shadow electron micrographs showed a persistence of a small, pepsin-sensitive terminal region in an amount consistent with that seen by electrophoresis. In vitro, this purified type V collagen formed thin fibrils with no apparent periodicity, while type I collagen fibrils had a broad distribution of large diameters. However, when type I collagen was mixed with increasing amounts of type V collagen a progressive and significant decrease in both the mean fibril diameter and the variance was observed for D periodic fibrils. The amino-terminal domain of the type V collagen molecule was required for this regulatory effect and in its absence little diameter reducing activity was observed. Electron microscopy using collagen type-specific monoclonal antibodies demonstrated that the fibrils formed were heterotypic, containing both collagen types I and V. These data indicate that the interaction of type V with type I collagen is one mechanism modulating fibril diameter and is at least partially responsible for the regulation of collagen fibril formation.