Human influenza viral infection in utero alters glial fibrillary acidic protein immunoreactivity in the developing brains of neonatal mice.

Epidemiological reports describe a strong association between prenatal human influenza viral infection and later development of schizophrenia. Postmodern human brain studies, however, indicate a lack of gliosis in schizophrenic brains presumably secondary to absence of glial cells during the second trimester viral infection in utero. We hypothesized that human influenza infection in day 9 pregnant mice would alter the expression of glial fibrillary acidic protein (GFAP, an important marker of gliosis, neuron migration, and reactive injury) in developing brains of postnatal days 0, 14 and 35 mice. Determination of cellular GFAP immunoreactivity (IR) expressed as cell density in cortex and hippocampus of control and experimental brains showed increases in GFAP-positive density in exposed cortical (P = 0.03 day 14 vs control) and hippocampal cells (P = 0.035 day 14, P = 0.034 day 35). Similarly, ependymal cell layer GFAP-IR cell counts showed increases with increasing brain age from day 0, to days 14 and 35 in infected groups (P = 0.037, day 14) vs controls. The GFAP-positive cells in prenatally exposed brains showed 'hypertrophy' and more stellate morphology. These results implicate a significant role of prenatal human influenza viral infection on subsequent gliosis, which persists throughout brain development in mice from birth to adolescence.

Programmed cell death of keratinocytes culminates in apoptotic secretion of a humectant upon secretagogue action of acetylcholine.

The programmed cell death of the stratified squamous epithelial cells comprising human epidermis culminates in abrupt transition of viable granular keratinocytes (KC) into dead corneocytes sloughed by the skin. The granular cell-corneocyte transition is associated with a loss in volume and dry cell weight but the mechanism for and biological significance of this form of keratinocyte apoptosis remain obscure. We show that terminally differentiated KC extrude into the intercellular spaces of living epidermis the cytoplasmic buds containing randomly congregated components of the cytosol as well as filaggrin, a precursor of the natural moisturizing factor. The discharge of secretory product is reminiscent of holocrine secretion, suggesting the term 'apoptotic secretion' for this novel, essential step in the process of cornification. The secretory product may become a part of the glycocalyx (a.k.a. 'intercellular cement substance' of epidermis) and serve as a humectant that counterbalances the osmotic pressure imposed by the natural moisturizing factor located in the stratum corneum comprised by corneocytes. The apoptotic secretion commences upon secretagouge action of acetylcholine which is synthesized and released by KC. A combination of a cholinergic nicotinic agonist and a muscarinic antagonist which increases intracellular calcium levels is required to trigger the apoptotic secretion. Analysis of the relative amounts of cholinergic enzymes and receptors expressed by KC capable of secretion and the pharmacological profiles of secretion regulation revealed an upward concentration gradient of free acetylcholine in epidermis which may provide for its unopposed secretagogue action via the m1 muscarinic and the alpha7, and alpha9 nicotinic receptor types expressed by KC at the latest stage of their development in the epidermis.

Ber-EP4-positive phenotype differentiates actinic keratosis from superficial basal cell carcinoma.

BACKGROUND: Well-defined histopathologic criteria exist to distinguish actinic keratosis (AK) from superficial basal cell carcinoma (BCC). A similar morphology of downwardly budding dysplastic keratinocytes may occur in both entities, creating potential for errors in diagnosis. A marker that could reliably distinguish these two lesions would overcome this difficulty in diagnosis. OBJECTIVE: To investigate whether Ber-EP4 staining is useful in distinguishing AK from superficial BCC, and to determine whether AK exhibits a cellular phenotype that is more consistent with BCC or squamous cell carcinoma (SCC). METHODS: We subjected tissue sections from superficial BCC, AK, and squamous intraepithelial neoplasia (SIN) demonstrating epidermal budding to immunohistochemical staining with monoclonal antibody Ber-EP4. RESULTS: Abnormal keratinocytes in all specimens of superficial BCC (5 of 5) were Ber-EP4 positive; all AK (10 of 10) and SIN (8 of 8) were Ber-EP4 negative. CONCLUSION: Ber-EP4 staining reliably distinguishes AK from superficial BCC. The lack of Ber-EP4 staining of AK supports the currently accepted pathogenetic dogma that SIN and SCC arise from AK, but BCC does not.

Venlafaxine and bupropion combination therapy in a case of treatment-resistant depression.

OBJECTIVE: To report the therapeutic efficacy of venlafaxine and bupropion in a patient with treatment-refractory major depression. CASE SUMMARY: A 21-year-old white woman with chronic and recurrent major depression presented with lack of response to several antidepressants. On examination, the patient exhibited neurovegetative signs of depression, guilt feelings, and suicidal ideation. The patient was administered venlafaxine 75 mg three times daily. The dose was titrated to 150 mg three times daily over the next month. Later bupropion was instituted up to 100 mg three times daily over a four-month period. The patient responded favorably to combination therapy and has remained free of depression for approximately 23 months. DISCUSSION: Venlafaxine and bupropion are antidepressant agents with unique pharmacologic profiles, each effective in the treatment of depression. Recent data indicate that combinations of selective serotonin-reuptake inhibitors and bupropion can convert partial response to full response in patients with treatment-resistant depression. We considered whether a combination of venlafaxine and bupropion would reduce the depressive symptoms of a patient who was unresponsive to various classes of psychotropic agents. Gradual administration of venlafaxine and bupropion acted synergistically to significantly reduce depressive symptoms (p < 0.002) and significantly increase social function (p < 0.002) over a period of eight months. CONCLUSIONS: To our knowledge this is the first report of successful combination therapy with venlafaxine and bupropion in treatment of chronic recurrent and refractory major depression.

Defective corticogenesis and reduction in Reelin immunoreactivity in cortex and hippocampus of prenatally infected neonatal mice.

Recent reports indicate an association between second trimester human influenza viral infection and later development of schizophrenia. Postmortem human brain studies also provide evidence for reduction in Reelin mRNA (an important secretory protein responsible for normal lamination of the brain) in schizophrenic brains. We hypothesized that human influenza infection in day 9 pregnant mice would alter the expression of reelin in day 0 neonatal brains. Prenatally-infected murine brains from postnatal day 0 showed significant reductions in reelin-positive cell counts in layer I of neocortex and other cortical and hippocampal layers when compared to controls. Whereas layer I Cajal-Retzius cells produced significantly less Reelin in infected animals, the same cells showed normal production of calretinin and nNOS when compared to control brains. Moreover, prenatal viral infection caused decreases in neocortical and hippocampal thickness. These results implicate a potential role of prenatal viral infection in causation of neuronal migration abnormalities via reduction in Reelin production in neonatal brains.

Differential expression of synaptosome-associated protein 25 kDa [SNAP-25] in hippocampi of neonatal mice following exposure to human influenza virus in utero.

We investigated the role of maternal exposure to human influenza virus [HI] in C57BL/6 mice on day 9 of pregnancy on the hippocampal expression of SNAP-25 in postnatal day 0 neonates, and compared them to sham-infected pups. The expression of SNAP-25 in infected neonates varied along the septotemporal axis of hippocampus and in various anatomic layers. Quantitative densitometric analysis of specific immunogold silver-enhanced SNAP-25 immunoreactivity [IR] showed increases of 40-347% over control in all septal-dorsal hippocampal layers except for the subplate layer. In mid septo-temporal hippocampus, SNAP-25 IR increased by 10-114% over control in all layers, except for the hippocampal plate, but the extent of this increase was smaller than in the dorsal-septal area. Finally,in temporal-ventral levels, SNAP-25 expression was reduced in all infected layers by 21-33% below control except for mild increases of 8.8 and 10% in subplate and hippocampal plate layers. Additionally, the infected SNAP-25 maximal density bin shifted to lower values dorsally and to higher values medially, with ventral maximal bins remaining unchanged when compared to controls. The differential expression of SNAP-25 in the hippocampi of infected neonates indicates a variable degree of vulnerability across the septo-temporal axis of hippocampus. It is surmised that while viral infection may induce excitotoxicity in the ventral hippocampus, it may cause reactive synapto-genesis in the medial and dorsal sectors of the developing brains of postnatal day 0 neonates.

Abnormal expression of epiligrin and alpha 6 beta 4 integrin in basal cell carcinoma.

BACKGROUND: Basal cell carcinoma is characterized by local invasion, and only rarely metastasizes. The role of the containing basement membrane (BM) in this tumor is unclear. Several BM components have been shown to be absent or significantly reduced in BM surrounding infiltrating tumor. OBJECTIVE: The purpose of this study is to examine the expression of epiligrin, a BM-associated glycoprotein, and the integrin chains alpha 3, alpha 6, beta 1, and beta 4 in the basement membranes surrounding basal cell carcinoma. METHODS: Samples were obtained from 20 patients with basal cell carcinomas and subjected to a standard avidin biotin complex/alkaline phosphatase immunohistochemical technique using a panel of antibodies. RESULTS: There was a consistent abnormality of expression of epiligrin, alpha 6, and beta 4. CONCLUSION: We propose that reduced expression of epiligrin is involved in the pathogenesis of the local invasion by tumor and that an altered integrin ratio in basal cell carcinoma enhances tumor spread.

Human influenza viral infection in utero increases nNOS expression in hippocampi of neonatal mice.

We investigated the role of maternal exposure to human influenza virus (HI) in C57BL/6 mice on day 9 of pregnancy on hippocampal expression of nNOS in day 0 neonates and compared that to sham-infected pups. Qualitative analysis using polyclonal antibody to nNOS showed overall increases in immunoreactivity (IR) in hippocampal and dentate layers of day 0 infected neonates when compared to sham-infected animals. These increases in nNOS immunoreactivity were pronounced in hippocampal plate, intermediate, molecular, subplate, and dentate areas. Quantitative analysis of specific immunogold silver-enhanced nNOS IR via densitometry showed nNOS IR increases of 26-71.6% in all layers, i.e., hippocampal plate (35.1%), dentate area (71.6%), molecular area (43.75%), subplate (45.7%), and intermediate zone (26%) in infected neonatal brains vs. controls. The changes in levels of nNOS expression in hippocampi of neonates born to mothers exposed to HI virus during the second trimester of pregnancy may reflect the potential for glutamatergic excitotoxicity via activation of NMDA receptors in the developing brains of these neonatal mice.

Anti-human epithelial antigen (Ber-EP4) helps define basal cell carcinoma masked by inflammation.

BACKGROUND: Anti-human epithelial antigen (Dako-Ber-EP4) is an antibody raised in mice that reacts with two glycoproteins of 34 and 49 kD. These glycoproteins are present on the cell surface and in the cytoplasm of basal cell carcinoma (BCC) cells, sweat glands, and some hair follicles in the skin. METHODS: We selected 27 BCCs (15 nodular, 11 morpheic/infiltrative, and one adenoid) and one trichoblastoma and performed rapid immunohistochemical studies with Ber-EP4 and a labeled streptavidin biotin alkaline phosphatase system. RESULTS: Twenty-seven of 27 BCCs and one of one trichoblastoma were positive for Ber-EP4. Thirteen of 27 BCCs stained with Ber-EP4 showed areas of BCC in dense inflammation that were better defined by the Ber-EP4 immunostain than by the H&E stain. In two cases persistent infiltrative BCC was found in the final Mohs margins while appearing negative with routine H&E. Several instances occurred where negative Ber-EP4 in inflammatory fields resulted in tissue sparing with the avoidance of a further Mohs (insurance) layer. CONCLUSION: In conclusion, we found mouse anti-human Ber-EP4 a useful and reliable marker for BCC. This antibody helps to locate latent BCC tumor in inflammatory Mohs margins.

Topical anesthetic agents in dermatologic surgery. A review.

BACKGROUND: The ideal topical anesthetic agent is one that provides 100% anesthesia in a short period of time, work on intact skin without systemic side effects, and invokes neither pain nor discomfort. The quest to find such an agent continues today. Because a topical anesthetic agent will induce anesthesia painlessly, the need for an effective agent is clear. This will serve to eliminate painful injections with lidocaine prior to many dermatologic procedures. OBJECTIVE: To provide a review of topical agents used in the past, to present products that are being used today, and to look to the future of topical anesthesia. CONCLUSIVE: During the last three decades a variety of methods have been employed to administer topical anesthesia. Presently, EMLA (eutectic mixture of local anesthetics) is the most often used method among practicing dermatologists. However, iontophoresis and the anesthetic patch are equally effective with a few notable advantages over EMLA. Liposomal agents show promise as we enter into a new millennium.

Nodular basal cell carcinoma in vivo vs in vitro. Establishment of pure cell cultures, cytomorphologic characteristics, ultrastructure, immunophenotype, biosynthetic activities, and generation of antisera.

BACKGROUND AND DESIGN: In this study we developed an in vitro model of nodular basal cell carcinoma (BCC). We obtained pure cultures of BCC cells and compared the morphologic characteristics, ultrastructure, immunophenotype, and behavior of cultured tumor cells with those of their in vivo counterparts. Tumors were excised from patients undergoing Mohs micrographic surgery. We established 69 primary cell cultures from 32 patients with nodular BCC. RESULTS: Three cell types grew in primary cultures: fibroblasts, normal-appearing keratinocytes, and cells with dual (spindle and epithelioid) morphologic characteristics. Contaminating fibroblasts were removed using 0.125% trypsin-0.02% edetic acid, and normal-appearing keratinocytes were cornified and eliminated by temporarily increasing the concentration of calcium in the growth medium. The cells with dual morphologic characteristics remained intact and exhibited relentless growth in pure cultures. That these seemingly immortal cell strains represent true nodular BCC was demonstrated by (1) their biphasic morphologic characteristics and very slow cell growth rate, (2) their capability for anchorage-independent growth in soft agar, (3) their ultrastructural similarities to freshly excised nodular BCC, (4) their ability to generate antibodies selectively labeling nodular BCC tumor nests in vivo, and (5) their immunophenotypic similarities to BCC in vivo on more than 20 different cell markers. CONCLUSIONS: This study provides a simple technique for establishing pure cell cultures of nodular BCC and describes extensively the in vitro parameters of tumor cell growth. The striking differences in behavior of cultured tumor cells in the presence or absence of normal-appearing keratinocytes suggest that normal human epidermal keratinocytes can suppress the growth of BCC cells.

Activation of keratinocyte nicotinic cholinergic receptors stimulates calcium influx and enhances cell differentiation.

Human epidermal keratinocytes synthesize, secrete, and degrade acetylcholine and use their cell-surface nicotinic and muscarinic cholinergic receptors to mediate the autocrine and paracrine effects of acetyl-choline. Because acetylcholine modulates transmembrane Ca2+ transport and intracellular metabolism in several types of cells, we hypothesized that cholinergic agents might have similar effects on keratinocytes. Nicotine increased in a concentration-dependent manner the amount of 45Ca2+ taken up by keratinocytes isolated from human neonatal fore-skins. This effect was abolished in the presence of the specific nicotinic antagonist mecamylamine, indicating that it was mediated by keratinocyte nicotinic acetylcholine receptor(s). The sequences encoding the alpha 5 and alpha 7 nicotinic receptor subunits were amplified from cDNA isolated from cultured keratinocytes. These subunits, as well as the alpha 3, beta 2, and beta 4 subunits previously found in keratinocytes, can be components of Ca(2+)-permeable nicotinic receptor channels. To learn how activation of keratinocyte nicotinic receptors affected the rate of cell differentiation, we measured the nicotinic cholinergic effects on the expression of differentiation markers by cultured keratinocytes. Long-term incubations with micromolar concentrations of nicotine markedly increased the number of cells forming cornified envelopes and the number of cells staining with antibodies to suprabasal keratin 10, transglutaminase type I, involucrin, and filaggrin. The increased production of these differentiation-associated proteins was verified by Western blotting. Because nicotinic cholinergic stimulation causes transmembrane Ca2+ transport into keratinocytes, and because changes in concentrations of intracellular Ca2+ are known to alter various keratinocyte functions, including differentiation, the subcellular mechanisms mediating the autocrine and paracrine actions of epidermal acetylcholine on keratinocytes may involve Ca2+ as a second messenger.

Bart's syndrome. Ultrastructure and genetic linkage.

BACKGROUND AND DESIGN: Bart's syndrome, originally described in a large family in 1966, consists of congenital localized absence of skin, blistering, and associated nail abnormalities. Since then, several descriptions of patients with similar clinical findings have suggested that this syndrome may represent any of the three subtypes of epidermolysis bullosa: epidermal, junctional, or dermal. Because no histologic or ultrastructural studies were done in Bart's kindred, and neither immunohistologic nor genetic linkage technology was available at that time, classification of the syndrome has been unclear. We report the findings of clinical, ultrastructural, immunohistologic, and genetic linkage studies of the original kindred and their descendants. We contacted original family members and their descendants by telephone and questionnaire. Skin biopsy specimens adjacent to blisters were obtained for ultrastructural and immunochemical analysis. Blood samples were drawn from affected members and their spouses and children for genetic linkage studies. RESULTS: The clinical findings seen in the descendants of the original family with Bart's syndrome were similar to those described in 1966. We did, however, detect persistence of blistering into adult life and mild atrophic scarring and milia formation at sites of blistering in some family members, a finding not noted in the original study. Hypertrophic scarring and albopapuloid lesions were not detected. Ultrastructural analysis of skin from affected family members showed poorly formed anchoring fibrils and cleavage below the lamina densa. Immunohistochemical staining localized type IV collagen at the roof of blistered skin. Staining for type VII collagen was found to have a normal distribution in nonblistered skin. Genetic linkage studies mapped the gene for the disease in this family to chromosome 3p at or near the site of the gene encoding type VII collagen. CONCLUSION: Bart's syndrome is a subtype of dominantly inherited dystrophic epidermolysis bullosa.

Keratinocyte muscarinic acetylcholine receptors: immunolocalization and partial characterization.

We have reported previously that human keratinocytes synthesize and secrete acetylcholine and that muscarinic cholinergic drugs have effects on keratinocyte proliferation, adhesion, and migration. This study defines the location of muscarinic acetylcholine receptors in human epidermis and describes some pharmacologic and molecular properties of these receptors. Confocal microscopy employing the anti-muscarinic receptor monoclonal antibody M35 visualized the receptors in the intercellular areas of normal human epidermis. Using immunoelectron microscopy, the receptors appeared to be attached to the keratinocyte plasma membranes. Functional, high-density (Bmax = 8.3 nmol/2 x 10(6) cells) and high-affinity (Kd = 21.5 nM) muscarinic receptors were demonstrated by saturable binding of the reversible radioligand [3H]quinuclidinyl benzilate to the surfaces of freshly isolated epidermal cells at 0 degrees C. Receptor proteins were separated by gel electrophoresis. An apparent isoelectric point of pH 4.3 was determined in immunoblots of sodium-cholate-solubilized receptors separated on isoelectric-focusing gels. Three protein bands, two at approximately 60 kDa and one at 95 kDa, were visualized in immunoblots of membrane-bound or solubilized receptors separated by sodium dodecylsulfate-polyacrylamide gel electrophoresis. The covalent, irreversible ligand [3H]propylbenzilylcholine mustard confirmed these results. Thus, human keratinocytes express a heterogeneous population of muscarinic cholinergic receptors. Because human keratinocytes also express nicotinic cholinergic receptors, endogenously secreted acetylcholine may control different biologic processes in these cells by activating different types of their cholinergic receptors.

Rapid cytokeratin stains enhance the sensitivity of Mohs micrographic surgery for squamous cell carcinoma.

BACKGROUND: Recurrence of squamous cell carcinoma (SCC) following Mohs micrographic surgery is uncommon. However, such cases do exist, presumably because of incomplete excision. Identification of single cells or small clumps of SCC tumor may be extremely difficult and can be compromised by inflammatory reaction. OBJECTIVE: The purpose of this study was to evaluate the benefits of incorporating rapid cytokeratin (CK) stains into Mohs technique. METHODS: Simple modification of standard immunoenzyme techniques allows keratin-specific staining to be achieved in less than 90 minutes on Mohs cryostat sections. We used the rapid labeled streptavidin biotin anticytokeratin method at the stage when no tumor was apparent by hematoxylin and eosin staining in 20 patients with large, aggressive, or recurrent invasive SCCs. RESULTS: In eight cases, single cells or small clumps of SCC tumor were identified utilizing AE-1 monoclonal antibody. These patients subsequently underwent further surgery, including wider tumor resection, superficial parotidectomy, or postoperative radiation therapy. CONCLUSION: The rapid CK antibody staining technique enhances the sensitivity of tumor identification in Mohs micrographic surgery, and should reduce tumor recurrence rates.

Rapid staining with carcinoembryonic antigen aids limited excision of extramammary Paget's disease treated by Mohs surgery.

Extensive extramammary Paget's (EMPD) disease of the perineum in a 68-year-old man was treated by Mohs surgery. To facilitate identification of involved tissue a rapid staining carcinoembryonic antigen was used. This technique proved a useful adjunct to conventional hemotoxylin-eosin (H&E) stains. It was especially useful in highlighting involvement in areas of marked dysplasia/artifact where discrimination is often difficult. It is recommended that such a technique offers considerable benefits over H&E staining when confronted by such tissue morphology.

Human keratinocytes synthesize, secrete, and degrade acetylcholine.

We previously reported that normal human keratinocytes express muscarinic receptors, and that acetylcholine induces attachment of these cells to each other. We have now studied the ability of human keratinocytes to synthesize, secrete, and degrade acetylcholine. To detect and localize the synthesizing enzyme choline acetyltransferase and degrading enzyme acetylcholinesterase, cultured cells and cryostat sections of normal human skin were pre-incubated with specific monoclonal antibodies and stained with an avidin-biotin complex/alkaline phosphatase. The choline acetyltransferase activity was assessed by the conversion of [3H]acetyl CoA to [3H]acetylcholine, and newly synthesized [3H]acetylcholine was detected using thin-layer chromatography. The acetylcholinesterase activity was measured spectrophotometrically. Both cholinergic enzymes were present in cultured keratinocytes, and in basal, spinous and granular epidermal cell layers. Choline acetyltransferase was visualized in the vicinity of cell nuclei, and acetylcholinesterase was observed in or near cell membranes. Newly synthesized acetylcholine was detected in both cell homogenates and culture supernatants. The estimated Vmax of the synthesis of labeled acetylcholine by homogenized keratinocytes was about 20 pmoles acetylcholine produced/mg protein/min at 37 degrees C. A single keratinocyte synthesized a mean of 2 x 10(-17) moles, and released 7 x 10(-19) moles acetylcholine per minute. Both cell homogenates and culture supernatants exhibited similar acetylcholinesterase activities indicating that human keratinocytes secrete acetylcholinesterase, too. Thus, we have demonstrated that normal human keratinocytes possess choline acetyltransferase and acetylcholinesterase, and synthesize, store, release, and degrade acetylcholine. Because human keratinocytes can also respond to acetylcholine, we believe that keratinocyte acetylcholine works in the epidermis as a local hormone.

Direct transfer of curettings to cryostat chuck in Mohs micrographic surgery.

In the routine handling of Mohs specimens the curetted first layer is integral to the process of tumor identification. Small specimens may be smeared inadvertently on filter paper or gauze or even lost between the operating room and laboratory. An efficient method for processing small curetted specimens has been devised that bypasses the use of a tissue tray. This method improves tissue morphology and saves time.