Induction of SLPI (ALP/HUSI-I) in epidermal keratinocytes.

Secretory leukocyte protease inhibitor (SLPI) is a small, cationic protein that is known to be constitutively expressed by several glandular epithelia. SLPI inhibits leukocyte-derived proteinases, has anti-HIV-1, antibacterial, and anti-fungal properties, and interferes with the induction of synthesis of proinflammatory mediators in monocytes and macrophages. We now report that at both the mRNA and the protein level, SLPI shows inducible expression in a nonglandular epithelium. A weak expression of SLPI was found in the stratum granulosum of adult normal human epidermis; however, in lesional psoriatic epidermis and in migrating keratinocytes of healing wounds, a strong cytoplasmic staining was seen in the suprabasal keratinocytes. Remarkably, in the dermis adjacent to SLPI-expressing keratinocytes, SLPI was found extracellularly associated with elastin fibers, whereas the dermis in normal skin was negative. In cell culture, SLPI was hardly expressed in monolayers of proliferating keratinocytes. Differentiating cultures with a phenotype of normal skin expressed low levels of SLPI, whereas cultures with a regenerative/psoriatic phenotype expressed high levels. Functional studies with recombinant SLPI indicated that its antibacterial spectrum and potency are distinct from other anti-microbial peptides such as lysozyme and defensins. In view of the multiple functions of SLPI and the inducibility, we propose that it acts as an important first line defence mechanism in cutaneous injury.

[D-penicillamine in treatment of scleroderma "en coup de sabre"]. / D-Penizillamin in der Behandlung von Sklerodermie "en coup de sabre".

A ten year old boy with linear erythema above the right eyebrow and a groove in the underlying skull bone is described. Histological examination of a biopsy revealed a spotty, periadnexal, perivascular, lymphocytic infiltrate. The papillary dermis was almost absent and the collagen fibres were slightly thickened. Based on the clinical appearance and the histology a diagnosis of linear scleroderma (en coup de sabre) was made. As the signs and symptoms were progressive, treatment with D-penicillamine was started. Subsequently the erythema disappeared. The shallow groove in the skull bone remained palpable. After thirteen months, treatment was stopped. The patient is currently free of signs and symptoms after a follow-up period of three and a half years. We feel that the strong clinical improvement is due to treatment with D-penicillamine rather than to the natural course of the disease.

Expression of SKALP/elafin during wound healing in human skin.

Skin-derived antileukoproteinase (SKALP), also known as elafin, is a proteinase inhibitor with specificity for polymorphonuclear leucocyte (PMN)- derived elastase and proteinase-3. SKALP is absent in normal human epidermis, but is strongly induced in inflammatory dermatoses such as psoriasis. SKALP is putatively involved in the regulation of cutaneous inflammation by inhibiting PMN derived proteinases. The aim of this study was to investigate SKALP expression and PMN infiltration during wound healing in human skin. This was examined in healing excisional wounds in normal skin and in impaired healing in various types of chronic venous ulcers. Tissues were analysed using immunohistochemistry and Northern blot analysis. Healing of excisional wounds was studied from day 0 to day 14. An influx of PMN was seen rapidly after wounding and was maximal between day 2 and 4 and then subsided. SKALP was induced within 48 h and was expressed in the suprabasal keratinocytes of the wound edge and the migrating epidermal sheet. SKALP expression was maximal on day 4 and was downregulated at the time of complete reepithelialization (7-14 days). In venous ulcers, PMN were abundant in the wound bed and scarce under the wound edge. SKALP was strongly expressed in the keratinocytes of the wound edge in all types of ulcers studied. In the wound bed, SKALP was not detectable. Our results suggest that SKALP plays a role in the acute, inflammatory phase of wound healing. From the kinetics and topology of SKALP expression we surmise that it negatively regulates PMN infiltration.

Tenascin expression during wound healing in human skin.

In adult human skin, the expression of the extracellular matrix glycoprotein tenascin is limited. Under hyperproliferative conditions such as psoriasis and epidermal tumours, dermal tenascin expression is strongly upregulated. The aim of this study was to investigate the pattern and kinetics of tenascin expression in human skin during wound healing and to address the question of whether keratinocytes can directly interact with tenascin during re-epithelialization. Tenascin expression was investigated in excisional wounds in normal human skin, in explants of normal human skin, and in chronic venous ulcers, using immunohistochemistry. No tenascin staining was found directly underneath the leading edge of the sheet of migrating keratinocytes in the excisional wounds and explants. In the excisional wounds and the ulcers, dermal tenascin was strongly upregulated in areas adjacent to hyperproliferative epidermis. These hyperproliferative areas are located approximately 10-50 cells behind the leading edge, as assessed by staining for the Ki-67 antigen and the proliferating cell nuclear antigen (PCNA). At the later stages of normal wound healing and in the chronic ulcers, tenascin was also detected in the wound bed. In these areas, the dermal-epidermal junction stained positive for laminin but was negative for heparan sulphate. The absence of the latter basement membrane component suggests that the formation of a new basement membrane is not completed in these wounds. These findings suggest that tenascin is not a substrate for migrating keratinocytes; that the rapid induction of tenascin expression in the papillary dermis during wound healing results from interaction with the hyperproliferative epidermis; and that in the later stages of wound healing, keratinocytes can potentially interact with tenascin in the wound bed, because the basement membrane of the neo-epidermis is incomplete.

Epidermal proliferation is not impaired in chronic venous ulcers.

In this study we have investigated epidermal growth and differentiation during wound healing in human skin. The studies were performed in excisional wounds in normal skin and in chronic venous ulcers. Tissues were analyzed by immunohistochemical staining for proliferation-associated nuclear antigens (PCNA and Ki-67 antigen) and cytokeratin 16. Healing of excisional wounds was studied from day 2 to 14. Recruitment of resting (G0) epidermal cells started within 2 days after wounding; the number of cycling cells was maximal at day 4 and continued to be increased (compared to baseline levels in normal skin) after wound closure (7-14 days). Cytokeratin 16, a proliferation-associated keratin, was induced within 48 h and was expressed in the suprabasal keratinocytes of the wound edge. Cytokeratin 16 expression was maximal at day 4 and was still present in the neo-epidermis after restoration of epidermal continuity (7-14 days). Surprisingly, in chronic venous ulcers, cycling cells were present in the wound edges of all stages of the leg ulcers studied. Both the number and localization of cycling cells were similar to those in normal wound healing. Cytokeratin 16 was strongly expressed in all these ulcers. Our in vivo data demonstrate that recruitment of G0-cells into the cell cycle is not impaired in venous ulcers, which suggests that epidermal proliferation is not a limiting factor in the healing process of chronic venous ulcers.

Age-related changes in wound healing.

Knowledge of the physiology of wound healing, in particular the recovery of the dermal and epidermal compartments and the co-ordination of these processes by the cytokine network, is of great importance to rational wound management. The individual components of the wound healing process have been studied using various in vitro and in vivo models, comparing young, adult and aged individuals. Many of the processes involved in wound healing are impaired in the elderly. However, in elderly patients not suffering from concomitant diseases, the rate of wound healing is normal or only slightly reduced. Various 'systemic factors' (endocrine and haematological diseases, nutritional deficiencies and medications) and 'regional disorders' (vascular and neural diseases) may impair wound healing. These complicating conditions occur more frequently in aged subjects. Failure of wound healing in the elderly is a chronic disabling condition, which occurs frequently in our society, requiring a major investment of medical care.

Professional organisations of allied health-disciplines assessments: a review of four disciplines.

Quality assurance developments in The Netherlands among physical therapists, speech-therapists, dietitians and occupational therapists in The Netherlands are discussed emphasising profession-dependent characteristics. A general introduction will precede a short outline of "the state of the art" of each discipline. Some experiences with the implementation of peer review among speech-therapists and a proposal to integrate quality of care concepts in educational curricula, will conclude this review.

Recent changes in the sociodemographic profile of sterilization acceptors in Nepal.

The records of 8056 Nepalese males and 9291 females sterilized from 1979-1983 were analyzed. Compared with data from previous studies (1970-1976), significant decreases occurred in the average age of female acceptors (30.6 vs. 33.1, P less than 0.01) and wives of male acceptors (28.4 vs. 31.7, P less than 0.01). Also, the average number of living children per couple decreased by one child (4.9-3.9, P less than 0.01) for male acceptors and 0.6 (4.8-4.2, P less than 0.01) for female acceptors. Finally in 83.2% of the cases, the operation was performed within 3 years of the last delivery.

PIP: The records of 8056 Nepalese males and 9291 females sterilized from 1979-1983 were analyzed. Compared with data from previous studies (1970-1976), significant decreases occurred in the average age of female acceptors (30.6 vs. 31.7, P0.01) and wives of male acceptors (28.4 vs. 31.7, P0.01). Also, the average number of living children per couple decreased by 1 child (4.9-3.9, P0.01) for male acceptors and 0.6 (4.8--4.2, P0.01) for female acceptors. Finally in 83.2% of the cases, the operation was performed within 3 years of the last delivery. Since 1970-1971 there has been a progressive rise in the % of sterilization acceptors referred by the health worker. For male acceptors there has been nearly a 3-fold increase. The vast majority of both male and female acceptors elected to have their operations within 2 years of the last delivery, regardless of the outcome. It appears that most couples time sterilization to coincide with the cessation of breastfeeding. Care must be taken to guard against the health worker being overly zealous in motivating and mobilizing potential voluntary sterilization contraception candidates.

Specific binding of the adenovirus terminal protein precursor-DNA polymerase complex to the origin of DNA replication.

Initiation of adenovirus DNA replication is dependent on a complex of the precursor of the terminal protein and the adenovirus-coded DNA polymerase (pTP-pol complex). This complex catalyzes the formation of a covalent linkage between dCMP and pTP in the presence of a functional origin of DNA replication residing in the terminal nucleotide sequence of adenovirus DNA. We have purified the pTP-pol complex of adenovirus type 5 and studied its binding to double-stranded DNA. Using DNA-cellulose chromatography it could be shown that the pTP-pol complex has a higher affinity for adenovirus DNA than for calf thymus or pBR322 DNA. From the differential binding of the pTP-pol complex to plasmids containing adenovirus terminal sequences with different deletions, it has been concluded that a sequence of 14 nucleotide pairs at positions 9-22 plays a crucial role in the binding of pTP-pol to adenovirus DNA. This region is conserved in the DNA's of all human adenovirus serotypes and is obviously an important structural element of the adenovirus origin of DNA replication. Comparative binding studies with adenovirus DNA polymerase and pTP-pol indicated that pTP is responsible for the binding. The nature of the binding of pTP-pol to the conserved sequence will be discussed.

Immunological characterization of the role of adenovirus terminal protein in viral DNA replication.

The function of the adenovirus-coded terminal protein and its precursor in viral DNA replication was studied by raising an antiserum against the adenovirus type 5 (Ad5) terminal protein isolated from virions. This antiserum reacted with both the terminal protein and its precursor as measured by a radioimmunoassay. In an in vitro DNA replication system employing nuclear extracts the addition of antiserum inhibits replication when a DNA-terminal protein complex from adenovirions is used as template. The replication of a 3.8% terminal fragment of the Ad2 genome with a protein-free origin (derived from the plasmid XD-7) is also inhibited by the antiserum. This observation confirms a role of the terminal protein precursor in DNA replication. The antiserum completely inhibited the formation of a covalent complex between the precursor terminal protein and dCMP, which is essential for initiation. A function of the terminal protein in the elongation reaction was shown by the inhibitory effect of antiserum on DNA chain elongation in isolated nuclei from Ad5-infected cells. Also in the in vitro DNA replication system employing nuclear extracts the elongation reaction is strongly reduced by addition of the antiserum. These results indicate that the terminal protein and/or its precursor are not only involved in initiation of DNA replication but also in DNA chain elongation.

Temperature-sensitive initiation and elongation of adenovirus DNA replication in vitro with nuclear extracts from H5ts36-, H5ts149-, and H5ts125-infected HeLa cells.

Adenovirus DNA replication was studied in vitro in nuclear extracts prepared from HeLa cells infected at the permissive temperature with H5ts125, H5ts36, or H5ts149, three DNA-negative mutants belonging to two different complementation groups. At the restrictive temperature, H5ts125 extracts, containing a thermolabile 72-kilodalton DNA-binding protein, enable the formation of an initiation complex between the 82-kilodalton terminal protein precursor (pTP) and dCTP, but further elongation of this complex is inhibited. Wild-type DNA-binding protein or a 47-kilodalton chymotryptic DNA-binding fragment can complement the mutant protein in the elongation reaction. No difference in heat inactivation was observed between wild-type extracts and H5ts36 or H5ts149 extracts when the replication of terminal XbaI fragments of adenovirus type 5 DNA-terminal protein complex was studied. In contrast, the formation of a pTP-dCMP initiation complex, as well as the partial elongation reaction up to nucleotide 26, were consistently more temperature sensitive in mutant extracts. The results suggest that the H5ts36/H5ts149 gene product is required for initiation of adenovirus type 5 DNA replication and that the 72-kilodalton DNA-binding protein functions early in elongation.

Replication of origin containing adenovirus DNA fragments that do not carry the terminal protein.

Nuclear extracts from adenovirus type 5 (Ad5) infected HeLa cells were used to study the template requirements for adenovirus DNA replication in vitro. When XbaI digested Ad5 DNA, containing the parental terminal protein (TP), was used as a template preferential synthesis of the terminal fragments was observed. The newly synthesized DNA was covalently bound to the 82 kD preterminal protein (pTP). Plasmid DNAs containing the Ad2 origin sequence or the Ad12 origin sequence with small deletions were analyzed for their capacity to support pTP-primed DNA replication. Circular plasmid DNAs were inactive. When plasmids were linearized to expose the adenovirus origin, both Ad2 and Ad12 TP-free fragments could support initiation and elongation similarly as Ad5 DNA-TP, although with lower efficiency. These observations indicate that the parental terminal protein is dispensable for initiation in vitro. The presence of 29 nucleotides ahead of the molecular end or a deletion of 14 base pairs extending into the conserved sequence (9-22) destroyed the template activity. DNA with a large deletion within the first 8 base pairs could still support replication while a small deletion could not. The results suggest that only G residues at a distance of 4-8 nucleotides from the start of the conserved sequence can be used as template during initiation of DNA replication.