Directed expression of keratin 16 to the progenitor basal cells of transgenic mouse skin delays skin maturation.

We previously hypothesized that the type I keratin 16 (K16) plays a role in the process of keratinocyte activation that occurs in response to skin injury (Paladini, R.D., K. Takahashi, N.S. Bravo, and P.A. Coulombe. 1996. J. Cell Biol. 132:381-397). To further examine its properties in vivo, the human K16 cDNA was constitutively expressed in the progenitor basal layer of transgenic mouse skin using the K14 gene promoter. Mice that express approximately as much K16 protein as endogenous K14 display a dramatic postnatal phenotype that consists of skin that is hyperkeratotic, scaly, and essentially devoid of fur. Histologically, the epidermis is thickened because of hyperproliferation of transgenic basal cells, whereas the hair follicles are decreased in number, poorly developed, and hypoproliferative. Microscopically, the transgenic keratinocytes are hypertrophic and feature an altered keratin filament network and decreased cell-cell adhesion. The phenotype normalizes at approximately 5 wk after birth. In contrast, control mice expressing a K16-K14 chimeric protein to comparable levels are normal. The character and temporal evolution of the phenotype in the K16 transgenic mice are reminiscent of the activated EGF receptor- mediated signaling pathway in skin. In fact, tyrosine phosphorylation of the EGF receptor is increased in the newborn skin of K16 transgenic mice. We conclude that expression of K16 can significantly alter the response of skin keratinocytes to signaling cues, a distinctive property likely resulting from its unique COOH-terminal tail domain.

The functional diversity of epidermal keratins revealed by the partial rescue of the keratin 14 null phenotype by keratin 16.

The type I epidermal keratins K14 and K16 are remarkably similar at the primary sequence level. While a structural function has been clearly defined for K14, we have proposed that a function of K16 may be to play a role in the process of keratinocyte activation that occurs after acute injury to stratified epithelia. To compare directly the functions of the two keratins we have targeted the expression of the human K16 cDNA to the progenitor basal layer of the epidermis of K14 null mice. Mice null for K14 blister extensively and die approximately 2 d after birth (Lloyd, C., Q.C. Yu, J. Cheng, K. Turksen, L. Degenstein, E. Hutton, and E. Fuchs. 1995. J. Cell Biol. 129:1329-1344). The skin of mice expressing K16 in the absence of K14 developed normally without evidence of blistering. However, as the mice aged they featured extensive alopecia, chronic epidermal ulcers in areas of frequent physical contact, and alterations in other stratified epithelia. Mice expressing a control K16-C14 cDNA also rescue the blistering phenotype of the K14 null mice with only a small percentage exhibiting minor alopecia. While K16 is capable of rescuing the blistering, phenotypic complementation in the resulting skin is incomplete due to the multiple age dependent anomalies. Despite their high sequence similarity, K16 and K14 are not functionally equivalent in the epidermis and other stratified epithelia and it is primarily the carboxy-terminal approximately 105 amino acids of K16 that define these differences.

Onset of re-epithelialization after skin injury correlates with a reorganization of keratin filaments in wound edge keratinocytes: defining a potential role for keratin 16.

Injury to stratified epithelia causes a strong induction of keratins 6 (K6) and 16 (K16) in post-mitotic keratinocytes located at the wound edge. We show that induction of K6 and K16 occurs within 6 h after injury to human epidermis. Their subsequent accumulation in keratinocytes correlates with the profound reorganization of keratin filaments from a pan-cytoplasmic distribution to one in which filaments are aggregated in a juxtanuclear location, opposite to the direction of cell migration. This filament reorganization coincides with additional cytoarchitectural changes and the onset of re-epithelialization after 18 h post-injury. By following the assembly of K6 and K16 in vitro and in cultured cells, we find that relative to K5 and K14, a well-characterized keratin pair that is constitutively expressed in epidermis, K6 and K16 polymerize into short 10-nm filaments that accumulate near the nucleus, a property arising from K16. Forced expression of human K16 in skin keratinocytes of transgenic mice causes a retraction of keratin filaments from the cell periphery, often in a polarized fashion. These results imply that K16 may not have a primary structural function akin to epidermal keratins. Rather, they suggest that in the context of epidermal wound healing, the function of K16 could be to promote a reorganization of the cytoplasmic array of keratin filaments, an event that precedes the onset of keratinocyte migration into the wound site.

Functional differences between keratins of stratified and simple epithelia.

Dividing populations of stratified and simple epithelial tissues express keratins 5 and 14, and keratins 8 and 18, respectively. It has been suggested that these keratins form a mechanical framework important to cellular integrity, since their absence gives rise to a blistering skin disorder in neonatal epidermis, and hemorrhaging within the embryonic liver. An unresolved fundamental issue is whether different keratins perform unique functions in epithelia. We now address this question using transgenic technology to express a K16-14 hybrid epidermal keratin transgene and a K18 simple epithelial keratin transgene in the epidermis of mice null for K14. Under conditions where the hybrid epidermal keratin restored a wild-type phenotype to newborn epidermis, K18 partially but not fully rescued. The explanation does not appear to reside in an inability of K18 to form 10-nm filaments with K5, which it does in vitro and in vivo. Rather, it appears that the keratin network formed between K5 and K18 is deficient in withstanding mechanical stress, leading to perturbations in the keratin network in regions of the skin that are subjected either to natural or to mechanically induced trauma. Taken together, these findings suggest that the loss of a type I epidermal keratin cannot be fully compensated by its counterpart of simple epithelial cells, and that in vivo, all keratins are not equivalent.

Autophosphorylation of protein kinase C at three separated regions of its primary sequence.

The major autophosphorylation sites of the rat beta II isozyme of protein kinase C were identified. The modified threonine and serine residues were found in the amino-terminal peptide, the carboxyl-terminal tail, and the hinge region between the regulatory lipid-binding domain and the catalytic kinase domain. Because this autophosphorylation follows an intrapeptide mechanism, extraordinary flexibility of the protein is necessary to phosphorylate the three regions. Comparison of the sequences surrounding the modified residues showed no obvious recognition motif nor any similarity to substrate phosphorylation sites, suggesting that proximity to the active site may be the primary criterion for their phosphorylation.

The spatial distribution of perseverations in neglect patients during a nonverbal fluency task depends on the integrity of the right putamen.

Deficient inhibitory control leading to perseverative behaviour is often observed in neglect patients. Previous studies investigating the relationship between response inhibition and visual attention have reported contradictory results: some studies found a linear relationship between neglect severity and perseverative behaviour whereas others could not replicate this result. The aim of the present study was to shed further light on the interplay between visual attention and response inhibition in neglect, and to investigate the neural underpinnings of this interplay. We propose the use of the Five-Point Test, a test commonly used to asses nonverbal fluency, as a novel approach in the context of neglect. In the Five-Point Test, participants are required to generate as many different designs as possible, by connecting dots within forty rectangles. We hypothesised that, because of its clear definition of perseverative errors, the Five-Point Test would accurately assess both visual attention as well as perseverative behaviour. We assessed 46 neglect patients with right-hemispheric stroke, and performed voxel-based lesion-symptom mapping (VLSM) to identify neural substrates of perseverative behaviour as well as the spatial distribution of perseverations. Our results showed that the Five-Point Test can reliably measure neglect and perseverative behaviour. We did not find any significant relationship between neglect severity and the frequency of perseverations. However, within the subgroup of neglect patients who displayed perseverative behaviour, the spatial distribution of perseverations significantly depended on the integrity of the right putamen. We discuss the putative role of the putamen as a potential subcortical hub to modulate the complex integration between visual attention and response inhibition processes.

Implicit associative priming in a patient with left visual neglect.

Recent studies have suggested that patients with visual neglect may process, even if without awareness, perceptual features of the neglected stimuli. The aim of the current study was to further investigate whether the ignored stimuli are processed at a deeper level. Findings from a patient with damage to the right hemisphere and with left visual neglect (no hemianopia) are reported. He showed an associative priming in the neglected space: response to a word in the right visual field was faster when the word was preceded by the brief presentation of an associated word in the neglected field. When obliged to orient attention to the left side of a computer screen, he was not able to detect the presence of, to read aloud, or to judge the lexical status and semantic content of left-sided stimuli. This patient was able to perform a covert post-perceptual processing of the neglected stimuli, but could not do so when explicitly requested.

Patient position for a synchronous cervicothoracoabdominal two-team esophagectomy.

Cervicothoracoabdominal and cervicoabdominal approach are routinely adopted for total or subtotal esophagectomy. We propose a modification of the Nanson's patient position to optimize sequential or simultaneous left cervicotomy, laparotomy, and eventual right thoracotomy with one or two surgical teams. This technique permits better control of the operative field for each phase of the procedure with coordinated operating of two surgical teams on the neck, abdomen, and chest.

[Treatment of chronic critical ischemia of the lower limbs with spinal cord electrostimulation]. / Il trattamento dell'ischemia critica cronica degli arti inferiori con elettrostimolazione midollare.

The treatment of Fontaine's third and fourth stage chronic critical lower limb ischemia can be considered a medical, social and economic problem. One current form of therapeutic intervention in some cases is medullary electrostimulation (SCS: spinal cord stimulator). This study looks at the period from January 1998 to December 1997 in which patients were selected for an etiological and symptomatological examination. The criteria established at the European Consensus Conference on Critical Leg Ischemia were employed to perform medullary electrostimulation. The entire procedure included a trial period with a temporary implant and if considered tolerable and effective, a permanent implant. One hundred sixty-four patients (117 male and 46 female, aged 41-93) affected by peripheral obstructive arteriopathy were examined. Etiological causes included atherosclerosis (70.7%), diabetes or other atherosclerotic diseases (25%), inflammatory arteriopathy (1.8%) and chronic renal failure under dialysis treatment (2.4%). The procedure was successful in 103 patients (62.8%) while unsuccessful in 61 (37.2%). The best results were obtained in patients at the Fontaine's 3rd stage in which the limb was saved in 72.2% of the patients and at the beginning of the 4th stage with a success rate of 62.7%. The advanced 4th stage had a success rate of only 42.4%. From an etiological point of view, the rate of limb preservation for atherosclerosis patients was 68.1%, in diabetics 56% and inflammatory diseases 33%. However, no positive results were obtained in patients with chronic renal failure.

Central venous catheter replacement in the ICU: new site versus guidewire exchange.

AIM: Catheter infection (central venous catheter, CVC-I) and catheter-related bacteremia (CRB) are of particular interest with ICU patients; more than 40-60% of them require a CVC. This prospective observational study was performed to determine if a second episode of catheterization and guidewire exchange was related to increased CRB and CVC-I rates in the ICU. METHODS: Over a period of 3 years, patients requiring a CVC, with catheter care, tip and peripheral blood cultures, were observed. RESULTS: A total of 898 non-tunneled CVCs were examined. The infection rates for 707 first-positioned CVCs were 4.3/1 000 catheter-day (c.d.) for CVC-I and 1.62 for CRB. Replacement was carried out for 191 CVCs: 7 of 103 CVCs inserted in a new site (4.81/1 000 c.d.) and 2 of 88 guidewire exchanged CVCs (1.75/1 000 c.d.) were infected; 2 replaced CVCs were related to CRB (1.38/1 000 c.d.). A cannulation time of over 7 days was related to a higher infection risk with its progressive reduction after the third week: the absolute risk increase was from 5.3 to 1.01 and the relative risk increased from 2.39 to 0.45 for CVC-I. CONCLUSION: Prolonged indwelling time is a significant risk factor for catheter-related infections; the second episode of cannulation and guidewire exchange did not present significant risk factors for catheter-related infections. A strict stable protocol for catheter insertion, care and proper treatment are necessary to reduce both the catheter-related infection rate and cost.

Procalcitonin, C-reactive protein, white blood cells and SOFA score in ICU: diagnosis and monitoring of sepsis.

AIM: To determine in critically ill patients the value of procalcitonin (PCT), C-reactive protein (CRP), sequential organ failure assessment (SOFA) score and white blood cell count in diagnosis and monitoring of sepsis. METHODS: Patients admitted to a medicosurgical intensive care unit in a prospective, observational study, were observed consecutively. According to ACCP/SCCM Consensus Conference definition were defined 4 groups: SEPSIS/SS (sepsis, severe sepsis, septic shock), SIRS, No-SIRS and TRAUMA. RESULTS: Two hundred and fifty five clinical events on a total of 1 826 observation days were observed: 111 SEPSIS/SS, 49 TRAUMA, 45 SIRS and 50 No-SIRS. ROC values, in the diagnosis of sepsis, were 0.88 for PCT, 0.74 for CRP, 0.8 for Sepsis score, 0.74 for SOFA, 0.62 for neu-throphils granulocytes (p<0.05). The best cut-off values in the diagnosis of sepsis were 0.47 ng/mL for PCT and 128 mg/L for CRP. PCT and SOFA were higher in septic shock than in severe sepsis and sepsis (p<0.05 in all cases). The maximum CRP level in SEPSIS/SS was reached only after 24-48 h of observation. Admission PCT value of TRAUMA patients whom evolving in septic complication was higher than patients with a favourable course: 3.4 ng/mL (range 2.63-12.71) vs 1.2 ng/mL (range 0.5-5.2) (p<0.05). TRAUMA patients with septic complications present an early and quick significant increase of PCT (p<0.05). CONCLUSIONS: PCT and CRP may be useful together with bacteriological data in sepsis diagnosis; PCT and SOFA closer correlate with the infection severity; PCT is the better parameter to estimate severity, prognosis or further course of the disease.

Protein and DNA-sequence homologies between the V3-loop of human immunodeficiency virus type I envelope protein gp120 and immunoglobulin variable regions.

We found that a common amino acid sequence motif exists between the V3-loop region of the human immunodeficiency virus type I envelope protein HIV gp120 and the human immunoglobulin heavy chain variable regions of subclass III (Ig VH-III). In the Ig VH-III sequences, the common motif overlaps with framework-1, complementarity-determining-region-1 and framework-2. In the homologous regions, the two groups of sequences also have a similar distribution of residue variability. On the DNA sequence level, the homology includes the conserved rearrangement signals of the VH-III genes, which lends support to the speculation that the V3 region of gp120 also may be involved in rearrangement processes.

Cloning and characterization of multiple human genes and cDNAs encoding highly related type II keratin 6 isoforms.

The human type II keratin 6 (K6; 56 kDa) is expressed in a heterogeneous array of epithelial tissues under normal conditions, but is better known for its strong induction in stratified epithelia that feature an enhanced cell proliferation rate or abnormal differentiation. Previous work has established the existence of two functional genes encoding K6 protein isoforms in the human genome, although only a partial cDNA clone is available for K6a, the dominant human K6 isoform in skin epithelial tissues (Tyner, A., and Fuchs, E. (1986) J. Cell Biol. 103, 1945-1955). We screened human genomic and skin cDNA libraries with probes derived from the K6b gene, and isolated clones containing the full-length gene and cDNA predicted to encode K6a. A thorough characterization of a large number of genomic (57) as well as cDNA (64) clones further revealed the existence of as many as six different human K6 protein isoforms that are highly related at the gene structure, nucleotide sequence, and predicted amino acid sequence levels. Based on the information accumulated to date we propose an evolutionary model in which the multiplicity of human K6 genes is explained by successive gene duplication events. We further demonstrate that K6a is clearly the dominant K6 isoform in skin tissue samples and cultured epithelial cell lines and that the various isoforms are differentially regulated within and between epithelial tissue types. Our findings have direct implications for an understanding of the regulation and function of K6 during hyperproliferation in stratified epithelia and the search for disease-causing mutations in K6 sequences in the human population.

Improved detection of homology in distantly related proteins: similarity of adducin with actin-binding proteins.

A novel and generally applicable method is described for the detection of homology in distantly related proteins using a new domain sequence database that contains over 20,000 protein sequence segments of known function. The use of the method is illustrated on distantly related domains shared by complement components C1S and C1R, calcium-dependent serine proteinase and bone morphogenetic protein 1. New homologies are shown between human adducin and the actin-binding domains of alfa-actinin and dystrophin.

cDNA cloning and bacterial expression of the human type I keratin 16.

The human type I keratin 16 is constitutively expressed in a number of complex epithelial tissues, including skin, but is better known for its induction under conditions favoring enhanced proliferation or abnormal differentiation, including wound healing, psoriasis, and cancer. We cloned the coding sequence of human K16 by applying a coupled reverse transcription-polymerase chain reaction procedure to mRNAs prepared from cultured human skin keratinocytes. We then expressed the human K16 coding sequence in E. coli and purified the solubilized protein by anion-exchange chromatography. The recombinant protein recovered behaves similarly to human K14 (a related acidic keratin) on the anion-exchanger, co-migrates with native human K16 on SDS-PAGE (M(r) 48 kD), and reacts with antisera directed against human K16. Based on the nucleotide sequence obtained and the properties of the corresponding recombinant protein, we conclude that we have cloned the coding portion of the human K16 cDNA. The sequence data obtained in this study is compared to earlier reports of the human K16 sequence, which are conflicting in many respects. The availability of K16 in a purified recombinant form will allow us to study how its properties may relate to its function during wound healing and in skin diseases.

Overexpression of human keratin 16 produces a distinct skin phenotype in transgenic mouse skin.

Human cytokeratin 16 (K16; 48 kDa) is constitutively expressed in postmitotic keratinocytes in a variety of stratified epithelial tissues, but it is best known for the marked enhancement of its expression in stratified squamous epithelia showing hyperproliferation or abnormal differentiation. Of particular interest to us, K16 is strongly induced at the wound edge after injury to the epidermis, and its accumulation correlates spatially and temporally with the onset of reepithelialization. To examine the properties of K16 in its natural cellular context, we introduced a wild-type human K16 gene into the germ line of transgenic mice. Several transgenic lines were established and characterized. Under most conditions, the human K16 transgene is regulated tissue specifically in the skin of transgenic mice. Animals that feature low levels of transgene expression are indistinguishable from controls during the first 6-8 months of life. In contrast, transgenic animals expressing the transgene at higher levels develop skin lesions at 1 week after birth, coinciding with the emergence of fur. At a cellular level, alterations begin with the reorganization of keratin filaments and are first seen at the level of the hair follicle outer root sheath (ORS), where K16 expression is known to occur constitutively. The lesions then progressively spread to involve the proximal epidermis, with which the ORS is contiguous. Elevated transgene expression is associated with a marked thickening of these two epithelia, along with altered keratinocyte cytoarchitecture and aberrant keratinization but no keratinocyte lysis. The implications of this phenotype for epithelial differentiation, human genodermatoses, and wound healing in skin are discussed.

A proline residue in the alpha-helical rod domain of type I keratin 16 destabilizes keratin heterotetramers.

The type I keratins 14 (K14) and 16 (K16) are distinct in their assembly properties and their expression pattern despite a high degree of sequence identity. Understanding K16 function and regulation is of interest, given its strong induction in keratinocytes located at the wound edge after injury to stratified epithelia. We reported previously that, compared with K14, K16 forms unstable heterotetramers with either K5 or K6 as the type II keratin pairing partner (Paladini, R. D., Takahashi, K., Bravo, N. S., and Coulombe, P. A. (1996) J. Cell Biol. 132, 381-397). We show here that yet another related type I keratin, K17, forms stable heterotetramers with a variety of type II keratins, further accentuating the unique nature of K16. Analysis of chimeric K14-K16 proteins in a heterotetramer formation assay indicated that the instability determinant resides in a 220-amino acid segment within the alpha-helical rod domain of K16. Site-directed mutagenesis revealed that Pro188, an amino acid residue located in subdomain 1B of the rod, accounts quantitatively for the instability of K16-containing heterotetramers under denaturing conditions. In vitro polymerization studies suggest that the presence of Pro188 correlates with a reduction in assembly efficiency. In addition to their implications for the stable conformation of the keratin heterotetramers, these findings suggest that the tetramer-forming properties of K16 may influence its partitioning between the soluble and polymer pools, and hence contribute to its regulation in epithelial cells under resting and wound repair conditions.

Liver transplantation in HBsAg-positive HBV-DNA--negative cirrhotics: immunoprophylaxis and long-term outcome.

The presence of a positive hepatitis B surface antigen (HBsAg) has been considered a highly questionable indication for orthotopic liver transplantation. We report our experience in the treatment of HBsAg-positive HBV-DNA-negative cirrhotics with liver transplantation, whether or not followed by passive prophylaxis with specific immunoglobulins. Of the 123 cirrhotics who received transplants at our institution since May 1986, 39 (31.7%) were HBsAg positive; of these, 1 was HBV-DNA positive, and 4 were hepatitis Be antigen (HBeAg) positive. Since April 1991, 25 HBsAg-positive HBV-DNA-negative cirrhotics have undergone an original protocol with the periodical intramuscular administration of 5,000 IU of specific immunoglobulins starting in the anhepatic phase and lasting for at least 1 year. There were no differences among cirrhotics in terms of operative mortality and long-term survival with respect to the presence of the HBsAg. Of the 35 HBsAg-positive HBV-DNA-negative patients having a follow-up of 1 month or longer, 12 (34.3%) developed HBsAg recurrence; of them, 4 (33.3%) had received a complete prophylaxis, whereas 8 (66.7%) had not. The recurrence rate was 80% (8 out of 10) in the group of patients who had not received the prophylaxis and 16% (4 out of 25) in the group who had received the prophylaxis (P = .0003). The actuarial recurrence rate in the treated patients was 20.2% and 20.2% after 1 and 3 years, respectively, whereas in the untreated group it was 60.0% and 70.0% (P < .01). The hazard of recurrence of treated patients was reduced to 24.9% compared with untreated patients. Liver transplantation can be performed in HBsAg-positive HBV DNA negative patients without an increase in the operative risk or a worsening of long-term results. Immunoglobulin prophylaxis seems to be effective in preventing hepatitis recurrence after transplantation.