Effect of polymer deposition method on thermoresponsive polymer films and resulting cellular behavior.

Poly(N-isopropyl acrylamide) or pNIPAM is a thermoresponsive polymer that is widely studied for use in bioengineering applications. The interest in this polymer lies in the polymer's unique capability to undergo a sharp property change near physiological temperature, which aids in the spontaneous release of biological cells from substrates. Currently, there are many methods for depositing pNIPAM onto substrates, including atom-transfer radical polymerization (ATRP) and electron beam ionization. Each method yields pNIPAM-coated substrates with different surface characteristics that can influence cell behavior. In this work, we compare two methods of pNIPAM deposition: plasma deposition and codeposition with a sol-gel. The resulting pNIPAM films were analyzed for use as substrates for mammalian cell culture based on surface characterization (XPS, ToF-SIMS, AFM, contact angles), cell attachment/detachment studies, and an analysis of exocytosis function using carbon-fiber microelectrode amperometry (CFMA). We find that although both methods are useful for the deposition of functional pNIPAM films, plasma deposition is much preferred for cell-sheet engineering applications because of the films' thermoresponse, minimal change in cell density, and maintenance of supported cell exocytosis function.

Assignment of two loci for autosomal dominant adolescent idiopathic scoliosis to chromosomes 9q31.2-q34.2 and 17q25.3-qtel.

BACKGROUND: Adolescent idiopathic scoliosis (AIS) is the most common form of spinal deformity, affecting up to 4% of children worldwide. Familial inheritance of AIS is now recognised and several potential candidate loci have been found. METHODS: We studied 25 multi-generation AIS families of British descent with at least 3 affected members in each family. A genomewide screen was performed using microsatellite markers spanning approximately 10-cM intervals throughout the genome. This analysis revealed linkage to several candidate chromosomal regions throughout the genome. Two-point linkage analysis was performed in all families to evaluate candidate loci. After identification of candidate loci, two-point linkage analysis was performed in the 10 families that segregated, to further refine disease intervals. RESULTS: Significant linkage was obtained in a total of 10 families: 8 families to the telomeric region of chromosome 9q, and 2 families to the telomeric region of 17q. A significant LOD score was detected at marker D9S2157 Z(max) = 3.64 ( theta= 0.0) in a four-generation family (SC32). Saturation mapping of the 9q region in family SC32 defined the critical disease interval to be flanked by markers D9S930 and D9S1818, spanning approximately 21 Mb at 9q31.2-q34.2. In addition, seven other families segregated with this locus on 9q. In two multi-generation families (SC36 and SC23) not segregating with the 9q locus, a maximum combined LOD score of Z(max) = 4.08 ( = 0.0) was obtained for marker AAT095 on 17q. Fine mapping of the 17q candidate region defined the AIS critical region to be distal to marker D17S1806, spanning approximately 3.2 Mb on chromosome 17q25.3-qtel. CONCLUSION: This study reports a common locus for AIS in the British population, mapping to a refined interval on chromosome 9q31.2-q34.2 and defines a novel AIS locus on chromosome 17q25.3-qtel.

The effects of cell culture parameters on cell release kinetics from thermoresponsive surfaces.

The thermoresponsive properties of poly(N-isopropyl acrylamide) (pNIPAM) have led to its wide use in bioengineering applications, including the reversible adhesion of mammalian cells. The groups performing this research have used different solutions to initiate cell release and have varied the temperature of the solution during detachment. To our knowledge, there has been no direct correlation between the solution identity or temperature on the efficiency of cell release from pNIPAM films. In this work, we present a study of the effect of the solution type and temperature used to initiate detachment on the time required to achieve 100% detachment of bovine aortic endothelial cells (BAECs) from pNIPAM. The pNIPAM films used in this work were obtained using a novel technique using a spin-coated solution containing pNIPAM (spNIPAM). We found that the fastest, most reliable release of cells occurred below the LCST of the polymer at 4 degrees C in serum free media (SFM). As it is sometimes desirable to stop cell metabolism at the time of detachment (e.g., to "freeze" protein expression prior to subsequent analysis), the use of extremely cold SFM would be ideal in such cases.

Estimation of carcass composition using rib dissection of calf-fed Holstein steers supplemented zilpaterol hydrochloride.

A serial harvest was conducted every 28 d from 254 to 534 d on feed (DOF) to quantify changes in growth and composition of calf-fed Holstein steers (n = 115, initial body weight (BW) = 449.2 ± 19.9 kg). One-half were supplemented with the ß-2 adrenergic agonist zilpaterol hydrochloride (ZH; 8.33 mg/kg 100% dry matter (DM) basis) during the final 20 d followed by a 3-d withdrawal prior to harvest; the remainder was fed a non-ZH control (CON) ration. Five steers were randomly selected and harvested after 226 DOF which served as a reference point for modeling purposes. Fabricated carcass soft tissue was ground, mixed, and subsampled for proximate analysis. Moreover, following the traditional method of rib dissection which includes the 9th, 10th, and 11th rib contained within the IMPS 103 primal, the relationship of carcass chemical composition to 9-10-11 rib composition was evaluated. Carcasses in this investigation had more (P < 0.01) separable lean, fat, ash, and moisture concomitant with less bone and ether extract than rib dissections. However, protein levels were similar (P = 0.27) between carcasses and rib dissections. Using regression procedures, models were constructed to describe the relationship of rib dissection (RD) composition including separable lean (RDSL), separable fat (RDSF), separable bone (RDSB), ether extract (RDEE), protein (RDP), moisture (RDM), and ash (RDA) with carcass composition. Carcass lean (CL), carcass fat (CF), and carcass bone (CB) were correlated (P < 0.01) with RDSL, RDSF, and RDSB with simple r values of 0.41, 0.71, and 0.50, respectively. Chemical composition of the rib and carcass, carcass ether extract (CEE), carcass protein (CP), carcass moisture (CM), and carcass ash (CA) were correlated (P ≤ 0.01) with simple r values of 0.75, 0.31, 0.66, and 0.37, respectively. Equations to predict carcass fatness from rib dissection variables and ZH supplementation status were only able to account for 50 and 56%, of the variability of CF and CEE, respectively. Overall, the relationships quantified and equations developed in this investigation do not support use of 9/10/11 rib dissection for estimation of carcass composition of calf-fed Holstein steers.

GM-CSF-deficient mice are susceptible to pulmonary group B streptococcal infection.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) gene-targeted mice (GM-/-) cleared group B streptococcus (GBS) from the lungs more slowly than wild-type mice. Expression of GM-CSF in the respiratory epithelium of GM-/- mice improved bacterial clearance to levels greater than that in wild-type GM+/+ mice. Acute aerosolization of GM-CSF to GM+/+ mice significantly enhanced clearance of GBS at 24 hours. GBS infection was associated with increased neutrophilic infiltration in lungs of GM-/- mice, while macrophage infiltrates predominated in wild-type mice, suggesting an abnormality in macrophage clearance of bacteria in the absence of GM-CSF. While phagocytosis of GBS was unaltered, production of superoxide radicals and hydrogen peroxide was markedly deficient in macrophages from GM-/- mice. Lipid peroxidation, assessed by measuring the isoprostane 8-iso-PGF2alpha, was decreased in the lungs of GM-/- mice. GM-CSF plays an important role in GBS clearance in vivo, mediated in part by its role in enhancing superoxide and hydrogen peroxide production and bacterial killing by alveolar macrophages.

Fabrication yields of serially harvested calf-fed Holstein steers fed zilpaterol hydrochloride.

Holstein steers ( = 110) were fed zilpaterol hydrochloride (ZH) for 0 or 20 d before slaughter during a 280-d serial harvest study. Cattle were harvested every 28 d beginning at 254 d on feed (DOF) and concluding at 534 DOF. After slaughter, carcasses were chilled for 48 h and then fabricated into boneless closely trimmed or denuded subprimals, lean trim, trimmable fat, and bone. Inclusion of ZH increased cold side weight (CSW) by 10.3 kg ( < 0.01; 212.7 vs. 202.4 kg [SEM 1.96]) and saleable yield by 10.4 kg ( < 0.01; 131.9 vs. 121.5 kg [SEM 1.16]) in calf-fed Holstein steer carcasses. Additionally, saleable yield as a percentage of CSW increased ( ≤ 0.01) by 2.19% (62.64 vs. 60.45% [SEM 0.22]) for cattle supplemented with ZH. Subprimal weights were heavier ( ≤ 0.05) from cattle that received ZH except for the bottom sirloin ball tip, back ribs, and outside skirt regardless of slaughter endpoint. Yield of top round, bottom round, and knuckle was increased ( ≤ 0.01) following ZH supplementation by 0.37, 0.24, and 0.18%, respectively. Yield of the top sirloin butt, strip loin, and tenderloin was increased ( ≤ 0.01) concurrent with ZH supplementation by 0.18, 0.11, and 0.09%, respectively. Regarding the rib primal, the rib eye roll tended ( = 0.08) to had increased yield (2.80 vs. 2.72% [SEM 0.03]) with ZH supplementation; both back ribs and blade meat exhibited increased ( ≤ 0.04) yields of 0.04%. Relative to the chuck primal, increased ( ≤ 0.03) yields of shoulder clod, pectoral meat, and mock tender were observed (0.13, 0.07, and 0.04%, respectively). Yield changes for subprimal brisket, plate, and flank were limited to increased ( < 0.01) proportion of flank steak and elephant ear (cutaneous trunci), 0.07 and 0.04%, respectively. Feeding duration notably altered ( ≤ 0.01) weights and percentages of all subprimals except the brisket. Saleable yield increased ( ≤ 0.01) by 0.192 kg/d with additional DOF. Moreover, trimmable fat and bone increased ( ≤ 0.01) by 0.146 and 0.050 kg/d, respectively. These data illustrate improved saleable meat yields for calf-fed Holstein steers supplemented with ZH and provide the beef industry knowledge of fabrication yield changes throughout a wide range of harvest endpoints.

Objective biometric measurements of calf-fed Holstein steers fed in confinement.

Understanding the maximum slaughter size for calf-fed Holstein steers based on hip-height has become a contemporary issue in the beef processing industry. Increased carcass size, in terms of both weight and length, has outpaced the ability of some abattoirs to handle the larger animals. Moreover, some abattoirs have begun rejecting animals that exceed 147.3 cm (58 inches) at the hip, creating a challenge for Holstein cattle feeders. The objective of this study was to quantify the skeletal growth rate of calf-fed Holstein steers fed in confinement. Hip-height of calf-fed Holstein steers ( ≤ 135) was measured every 28 d from 226 to 422 d on feed. Hip-height was a dependent variable modeled via linear regression procedures utilizing days of age and BW as independent variables. Additionally, logistic regression was used to estimate the probability of a steer exceeding a hip-height of 147.3 cm (58 inches) from independent variables of days of age and BW. The linear relationship of BW to hip-height had an adjusted value of 0.7112 (Hip-height, cm = [0.0593 × BW, kg] + 109.00) and on average the calf-fed Holstein steers grew 1.0 cm for each 16.9 kg of BW gain during the finishing phase. The 10%, 50%, and 90% probability of a steer exceeding 147.3 cm (58 inches) of hip-height was achieved at 563, 653, and 743 kg of BW, respectively. The linear relationship of days of age to hip-height had an adjusted value of 0.6687 (Hip-height, cm = [0.0937 × days of age] + 104.4) and the calf-fed Holstein steers grew 1.0 cm for each 10.7 d of age during the finishing phase. The 10%, 50%, and 90% probability of a steer exceeding 147.3 cm (58 inches) of hip-height was estimated to occur at 408, 459, and 510 d of age, respectively. Knowledge of Holstein steer growth rate in relation to BW and age may allow for more accurate sorting to prevent oversized cattle arriving at the abattoir and subsequent discounts or being rejected for slaughter.

Cytoplasmic localization of the oncogenic protein Ski in human cutaneous melanomas in vivo: functional implications for transforming growth factor beta signaling.

The oncogenic protein Ski associates with Smad proteins and counteracts their activation of gene expression and growth inhibition in response to transforming growth factor beta (TGF-beta). Here we show that Ski protein levels are increased in all 44 human melanoma tumor tissues analyzed in vivo. In addition, Ski subcellular localization changes from nuclear, in preinvasive melanomas (melanomas in situ), to nuclear and cytoplasmic in primary invasive and metastatic melanomas. Furthermore, Ski/Smad association in the cytoplasm seems to prevent Smad3 nuclear translocation in response to TGF-beta. The biological significance of Ski overexpression in melanomas was established by showing that down-regulation of Ski levels, by antisense Ski vectors, restored TGF-beta-mediated growth inhibition. Such inhibition is apparently mediated by up-regulation of the cyclin-dependent kinase-I p21(Waf-1) and inhibition of cyclin-dependent kinase 2 activity. Our results suggest that high levels of Ski in human melanomas produce a disruption of TGF-beta signaling phenotypically similar to that in cells harboring mutations in TGF-beta receptors or Smad proteins, and this may represent a significant event in the progression of melanomas in vivo.

Loss of expression of the p16/cyclin-dependent kinase inhibitor 2 tumor suppressor gene in melanocytic lesions correlates with invasive stage of tumor progression.

Sporadic and familial malignant melanoma susceptibility has been linked to defects in the chromosomal region 9p21. Recently, a putative 9p21 tumor suppressor gene, the cyclin dependent kinase inhibitor 2 (CDKN2) or p16 gene, has been shown to be deleted, mutated, or rearranged in a high percentage of sporadic melanoma cell lines, as well as mutated in the germline of a proportion of familial melanoma patients. CDKN2 encodes a M(r) 16,000 protein (p16) that plays a key role in cell cycle control by binding to the cyclin-dependent kinase 4 enzyme and inhibiting its ability to phosphorylate critical substrates necessary for transition past the G1 phase of the cell cycle. Thus, mutations or deletions of the CDKN2 gene could result in abnormal proliferation via defective cell cycle control. The correlation of 9p21 cytogenetic and molecular alterations with the clinical stages of melanoma progression suggests that dysfunction of a gene within this chromosomal region is critical to the evolution of melanoma. However, it remains unclear whether this gene is the CDKN2 gene. If so, then loss of p16 is potentially an initiating or early event in melanoma progression. To address the issues of what is the potential involvement of the CDKN2 gene in sporadic melanoma and precisely when during the clinically evident stages of melanoma progression defects in CDKN2 occur, we have evaluated by immunohistochemistry the expression of p16 protein in 103 melanocytic lesions representing all stages in the progression of melanoma. Our results suggest that loss of p16 protein expression is (a) not necessary for tumor initiation in malignant melanoma because all melanomas in situ and the majority of primary invasive melanomas retain expression of this protein; and (b) potentially more related to invasiveness or the ability to metastasize, because 52% of primary invasive tumors and 72% of metastatic lesions show partial or complete loss of expression of p16.

Byproduct yields of serially harvested calf-fed Holstein steers fed zilpaterol hydrochloride.

A 2 × 11 factorial treatment structure was applied in a completely randomized experimental design to investigate differences in noncarcass tissue among serially harvested Holstein steers. Steers ( = 110) were randomly assigned to 1 of 2 dietary treatments: a ration supplemented with zilpaterol hydrochloride (ZH) fed at a rate of 8.3 mg/kg DM for 20 d followed by a 3-d withdrawal or a control ration with no ZH included in the diet. Within treatment, steers were assigned to harvest groups of 254, 282, 310, 338, 366, 394, 422, 450, 478, 506, or 534 d on feed (DOF) prior to initiation of the trial. Cattle fed ZH realized an empty BW (EBW) increase ( ≤ 0.03) of 2.8% (644.2 vs. 626.4 kg [SEM 5.4]) and a HCW increase of 5.0% (429.1 vs. 408.4 kg [SEM 4.0]) with a concomitant 12% reduction (45.1 vs. 51.2 kg [SEM 3.1]) in gastrointestinal contents and 2.1 percentage unit increase in dressed carcass yield (62.1 vs. 60.0% [SEM 0.01]). Additionally, ZH supplementation decreased (P ≤ 0.03) the absolute weight of the liver and kidneys by 0.3 and 0.1 kg, respectively. When noncarcass components were expressed on an empty body basis (g/kg EBW), reductions ( ≤ 0.01) in the limbs (18.8 vs. 19.5 g/kg EBW [SEM 0.1]), hide (81.1 vs. 78.1 g/kg EBW [SEM 0.7]), liver (14.2 vs. 13.2 g/kg EBW [SEM 0.2]), kidneys (2.6 vs. 2.3 g/kg EBW [SEM 0.04]), small and large intestines (74.9 vs. 69.6 g/kg EBW [SEM 1.2]), and gastrointestinal tract (119.8 vs. 113.4 g/kg EBW [SEM 1.3]) were observed with ZH supplementation. Additionally, there was a tendency ( = 0.07) for the proportion of total offal to be reduced (253.2 vs. 247.4 g/kg EBW [SEM 2.5]) with ZH supplementation. Empty BW and HCW linearly increased ( < 0.01) by 1.16 and 0.758 kg/d ( < 0.01), respectively, with additional DOF. The weight of the liver and intestines linearly increased ( < 0.01) by 0.007 and 0.133 kg/d ( < 0.01), respectively, with additional DOF. These data indicate the magnitude of change in noncarcass tissues that can be expected when calf-fed Holstein steers are supplemented with ZH.

The effect of days on feed and zilpaterol hydrochloride supplementation on feeding behavior and live growth performance of Holstein steers.

This experiment was designed to study the effect of days on feed (d 225-533) and zilpaterol hydrochloride (ZH) supplementation on Holstein steer ( = 110) performance and feeding behavior as part of a serial slaughter trial. Steers were randomly assigned to 1 of 11 harvest groups with 10 steers ( = 5 control and = 5 ZH; ZH at 8.33 mg/kg diet) harvested each 28 d. Steers were weighed every 28 d (d 225, 253, 281, 309, 337, 365, 393, 421, 449, 477, 505, and 533); individual daily meal consumption data for each steer were recorded using GrowSafe technology. In the pretreatment period, dry matter intake expressed a negative quadratic relationship with days on feed (DOF) {DMI = -5.7120 + (0.08370 x DOF)- (0.00011 x DOF); Adj. = 0.2574; RMSE = 0.25 75; 0.01}. A linear increase in BW ( < 0.01) occurred during the pretreatment 308 d period from 466 to 844 kg, {BWend = 137.61 + (1.4740 x DOF); Adj. = 0.8819; RMSE = 37.06; < 0.01}, whereas ADG and G:F decreased linearly. Dry matter intake per meal exhibited a quadratic relationship over days on feed and peaked ( < 0.01) during d 365 to 392 at 1.065 kg coinciding with the highest numerical daily DMI (11.19 kg). Daily consumption visit duration differed ( < 0.01) during the 308 d period, with a low of 52.29 min (d 337-364) and a high of 55.59 min (d 365-392). Consumption rate peaked at 714 g/min (d 337-364) and exhibited a quadratic relationship to DOF. The difference ( < 0.04) in DMI between control and ZH treated cattle across all 11 harvest groups averaged 0.575 kg. Moreover, ZH treatment resulted in decreased ( 0.01) DMI per meal event of 0.093 kg. Gain to feed tended to improve ( = 0.06) with ZH treatment by 0.017 kg gain per kg feed relative to the control cattle. Daily bunk, consumption, and meal visit durations were influenced by ZH during the 20 d treatment period ( = 0.01); the average difference between control and ZH supplemented cattle over the 308 d trial was 9.09, 8.71, and 11.39 min per d, respectively. The data collected in this trial indicate live growth performance and feeding behavior were impacted by both DOF and ZH supplementation.

Carcass grading characteristics of serially harvested calf-fed Holstein steers fed zilpaterol hydrochloride.

Serial harvests were conducted using Holstein steers ( = 110) fed zilpaterol hydrochloride (ZH) for 0 or 20 d prior to harvest. Steers were harvested in 28-d increments beginning at 254 d on feed (DOF) and ending at 534 DOF. After harvest and a 36-h chill period, carcasses were evaluated using grading methods standard for the United States (USDA), Canada (Canadian Beef Grading Association [CBGA]), and Japan (Japanese Meat Grading Agency [JMGA]). No ZH treatment differences ( = 0.81) were detected for 12th-rib fat thickness; however, additional DOF resulted in a daily linear increase ( < 0.01) of 12th-rib fat thickness by 0.004 cm/d. Longissimus muscle area was increased ( < 0.01) by 8.7 cm with ZH supplementation and linearly increased ( < 0.01) 0.08 cm2/d with additional DOF. Calculated USDA yield grade (YG) decreased ( < 0.01) 0.33 units due to ZH treatment and linearly increased ( < 0.01) 0.009 units/d. Steers supplemented with ZH exhibited increased ( < 0.01) CGBA LM width; however, no difference ( = 0.37) was detected in CGBA LM length. No ZH treatment differences ( = 0.64) were observed for CBGA fat class; however, CGBA fat class linearly increased ( < 0.01) by 0.01 units/d. No ZH differences ( ≥ 0.17) were detected for the CBGA estimated lean percentage or YG equations. Evaluation for JMGA occurs at the sixth and seventh rib interface; LM area was 4.6 cm2 greater ( = 0.02) for cattle supplemented with ZH and linearly increased ( < 0.01) by 0.07 cm2/d with additional DOF. Subcutaneous fat thickness was not different among ZH treatments ( = 0.10) but linearly ( < 0.04) increased ( < 0.01) by 0.005 cm/d with additional DOF using the JMGA grading method. No difference ( ≥ 0.21) was calculated between ZH treatments or DOF for JMGA estimated yield. No ZH treatment differences ( = 0.85) were detected in USDA marbling score; however, marbling linearly increased ( < 0.01) 0.07 units/d. These data illustrate the impact of ZH and increasing DOF on economically important carcass grading outcomes used in the USDA, CBGA, and JMGA grading programs.

Adenovirus-mediated granulocyte-macrophage colony-stimulating factor improves lung pathology of pulmonary alveolar proteinosis in granulocyte-macrophage colony-stimulating factor-deficient mice.

Mutation of the granulocyte-macrophage colony-stimulating factor (GM-CSF) gene by homologous recombination causes progressive pulmonary alveolar proteinosis (PAP) in GM-CSF-deficient mice (GM-/-). The present study tested whether adenovirus-mediated expression of GM-CSF alters the progression of PAP in GM-/- mice. Adult mice were pretreated with an anti-T cell receptor (TCR) antibody to block T cell-mediated immune response, followed by intratracheal instillation of deltaE1-E3 replication-deficient adenovirus expressing mouse GM-CSF (Av1mGM). Mice were killed 1, 3, and 5 weeks after treatment to assess lungs for GM-CSF, surfactant protein B (SP-B), alveolar macrophage maturation, and type II cell proliferation. GM-CSF was detected in BAL fluid from GM-/- mice 1 week after Av1mGM treatment, and GM-CSF mRNA was detected by RT-PCR through 5 weeks. Five weeks after Av1mGM treatment, PAP was improved and SP-B decreased as assessed by ELISA and immunostaining. Increased numbers of alveolar macrophages stained with alpha-naphthyl acetate esterase (alpha-NAE) following treatment with Av1mGM. Local expression of GM-CSF with a recombinant adenovirus ameliorated PAP in the GM-/- mice in association with enhanced maturation of alveolar macrophages.

Characterization of protein kinases in mitotic and meiotic cell extracts.

A number of protein kinases have been separated and identified in extracts from mitotic and interphase culture cells and from mature and immature amphibian oocytes using nondenaturing polyacrylamide gel electrophoresis followed by in situ phosphorylation assays. Certain of these protein kinase activities appear to correlate with the biological activity of extracts, assayed by their ability to induce meiotic maturation following injection into Xenopus oocytes. These results are consistent with the notion that protein phosphorylation/dephosphorylation may be integral to the mechanisms of both nuclear membrane breakdown and chromosome condensation, events common and distinctive to mitosis and meiosis.

Comparative changes in radial-bone density of elderly female lacto-ovovegetarians and omnivores.

A 5-y prospective study of the changes in radial-bone mineral density (BMD) of elderly white women (mean age, 81 y) living in four residential communities, including 49 Seventh-day Adventist lacto-ovovegetarians and 140 omnivores, was undertaken to determine the potential effects of usual dietary calcium in preventing the loss of BMD, measured by single-photon absorptiometry, at two radial sites. Changes in BMD and other variables from baseline (1983) to follow-up (1988) were: 1) mean calcium intakes in 1988 of 996 mg/d for omnivores and 733 mg/d for lacto-ovovegetarians changed little from 1983, 2) all women lost BMD (P < 0.05) over the 5 y period, 3) the annual BMD loss rates were approximately 1% at each site, 4) BMD loss was independent of calcium intake, 5) BMD loss rates were similar in both lacto-ovovegetarians and omnivores, and 6) the greater the loss of lean body mass, the greater the BMD loss (P < 0.05).

Differential expression of the intermediate filament peripherin in cutaneous neural lesions and neurotized melanocytic nevi.

Peripherin is an intermediate filament involved in growth and development of the peripheral nervous system, and is produced by neurons and the beta cells of the islets of Langerhans. Recently, malignant melanomas and some melanocytic nevi have been shown to express peripherin. It is unknown if Schwann cells, also derived from the neural crest, express peripherin. Expression of peripherin was evaluated by immunohistochemistry in cutaneous lesions characterized by a prominent Schwann cell component including 26 neurofibromas (NF), 10 schwannomas (SCH), seven granular cell tumors, and five palisaded encapsulated neuromas (PEN); 13 neurotized melanocytic nevi (NMN) also were evaluated because these lesions contain Wagner-Meissnerlike structures and type C nevus cells, which exhibit a "schwannian" phenotype. Peripherin was detected in the axons of normal peripheral nerves. NF and PEN contained numerous axons dispersed throughout the lesions, whereas only scattered small nerves were seen in GCT. In SCH, only rare axons were labeled, mostly at the periphery of the lesions. All other cells in these four types of lesions, therefore including Schwann cells, were not labeled. In most NMN, labeled axons were identified within the lesions. In a few cases, rare epithelioid melanocytes within the superficial portions of the nevi were labeled. The Wagner-Meissnerlike structures and type C nevus cells (schwannian) were not labeled in any lesion; however, numerous labeled axons invested these areas. Because there are different relative numbers of peripherin-labeled axons throughout NF, PEN, some nevi, and SCH, analysis of peripherin expression may be helpful in the diagnosis of these lesions. Neurons and some epithelioid melanocytes, in contrast to type C nevus cells and Schwann cells of NF and SCH, express peripherin, providing further evidence for a transition from a more neuronal to a more schwannian phenotype during the normal maturation sequence of melanocytes in nevi.

Primary cutaneous immunocytoma. A B-cell lymphoma that can easily be mistaken for cutaneous lymphoid hyperplasia.

It has been estimated that immunocytomas comprise roughly 2% of all cutaneous lymphomas. We studied five patients with primary cutaneous immunocytomas who presented with cutaneous nodules or plaques. Many of the infiltrates were "top-heavy" and polymorphous with admixed eosinophils, macrophages, lymphoid follicles, and non-neoplastic lymphocytes. Other potentially confusing findings were one case each of spongiotic dermatitis and leukocytoclastic vasculitis. The neoplastic cells were often situated at the peripheries of nodules and ranged from those with nuclei that resembled small lymphocytes to others that resembled immunoblasts. Most had eccentrically placed nuclei and fan-shaped cytoplasm. Monotypic kappa-light chain was found in all five cases, accompanied by gamma-heavy chain in two cases, and mu-heavy chain in one. In situ hybridization detected only kappa-mRNA in the four cases that yielded technically satisfactory results. The neoplastic cells did not express the B-cell antigen CD20; T-cells formed the centers of many nodules. Inappropriate staining for CD43 was evident in the neoplastic cells of one case. Because of reports of immunocytomas complicating acrodermatitis chronica atrophicans, we stained sections with an antiserum to Borrelia burgdorferi, which did not detect that organism. In situ hybridization did not detect EBER-1 RNA of the Epstein-Barr virus, which can be present in immunocytomas in immunocompromised patients. One patient died of disease after failing chemotherapy; another is alive with disseminated disease, and three are in remission following excision of lesions alone in two patients and chemotherapy in one patient who had relapsed following both excision and radiation therapy.

Immunocytochemical identification of Rochalimaea henselae in bacillary (epithelioid) angiomatosis, parenchymal bacillary peliosis, and persistent fever with bacteremia.

We report the immunocytochemical identification of Rochalimaea henselae, a newly recognized fastidious, Gram-negative, Warthin-Starry-positive organism, as the common pathogen in bacillary angiomatosis (BA), bacillary peliosis (BP) of the liver and spleen, and persistent fever with bacteremia in immunocompromised patients. Immunogenic proteins of the R. henselae strain isolated from the blood of a febrile immunocompromised patient with BP of the liver were used to produce primary immune serum in rabbits. Using immunocytochemical procedures, the polyclonal antiserum reacted strongly not only with the immunizing strain of the bacteria, but also with other blood isolates of R. henselae (five cases) from both immunocompromised and immunocompetent patients and with the organisms present in the tissue lesions of cutaneous BA (five cases) and BP of the liver (two cases) and spleen (one case). The blood isolates and BA and BP tissue samples were obtained from widely separated geographic areas. The antiserum was weakly cross-reactive with cultures of Rochalimaea quintana, an organism closely related to R. henselae, but this reactivity was eliminated by specific adsorption. The antiserum did not cross-react with the Warthin-Starry-positive organisms associated with cat scratch disease (Afipia felis), syphilis (Treponema pallidum), Lyme disease (Borrelia burgdorferi) or chronic active gastritis (Helicobacter pylori). Likewise, the antiserum did not identify organisms in eight cases of Kaposi's sarcoma, a disorder of immunocompromised patients that is clinically similar to BA. Further studies are needed to determine the prevalence of this newly recognized organism as well as its possible involvement in other angioproliferative diseases.

Desmoplastic (sclerotic) nevus: an underrecognized entity that resembles dermatofibroma and desmoplastic melanoma.

Desmoplastic (sclerotic) nevus, a benign melanocytic neoplasm characterized by predominantly spindle-shaped nevus cells within a fibrotic stroma, can be confused with fibrous lesions and other melanocytic proliferations, including desmoplastic melanoma. We compared the histologic and immunohistochemical features of 16 desmoplastic nevi, nine desmoplastic melanomas, four hypopigmented blue nevi, and six dermatofibromas. The similarities between desmoplastic nevi and dermatofibromas included epidermal hyperplasia (12 of 16), presence of keloidal collagen (15 of 16), hypercellularity (16 of 16), and increased numbers of factor XIIIa-positive dendritic cells (12 of 12). The absence of adnexal induction (0 of 16), the rarity of lesions with multinucleated cells (3 of 16) or epidermal hyperpigmentation (2 of 16), and the presence of S-100 immunoreactivity (16 of 16) and melanocytic proliferation (9 of 16) helped differentiate desmoplastic nevi from dermatofibromas. The similarities between desmoplastic nevi and desmoplastic melanomas included the presence of atypical cells (16 of 16) and HMB-45 expression in the superficial portion of the lesions (11 of 16). The infrequent location on the head or neck (1 of 16), the absence of mitotic figures (0 of 16), a significantly lower number of Ki-67-reactive cells, and a decrease in HMB-45 expression in the deep area of the lesions (8 of 11) helped distinguish desmoplastic nevi from desmoplastic melanoma. Desmoplastic nevi had overlapping features with hypopigmented blue nevi, but features tending to favor the latter included a predominance of ovoid nuclei, higher numbers of atypical cells, and homogeneous staining with HMB-45. We conclude that a combination of histologic and immunohistochemical criteria facilitates the reliable diagnosis of desmoplastic nevus from its simulators.

Complete one-hour immunocytochemistry based on capillary action.

We describe a less than one-hour manual method for immunocytochemical analyses of B5 or formalin-fixed, paraffin-embedded tissue sections. The method employs capillary action to sequentially apply, incubate and remove liquid reagents from apposed pairs of up to 20 glass microscope slides and allows for simultaneous immunocytochemical analyses of as many as 10 different antigens. The method described here uses a) positively charged glass slides to rapidly immobilize tissue sections; b) rapid deparaffinization techniques; c) multipurpose reagents; d) ethanol-enriched buffer washes to improve capillary action and reduce nonspecific background; e) a single broad spectrum streptavidin-peroxidase or streptavidin-alkaline phosphatase detection system that identifies most primary monoclonal and polyclonal antibodies; and f) specific immunocytochemical signal amplification by cyclic chromogen enhancement.