Novel proteases with unusual specificities.

This review describes specific proteases that have been implicated in several interesting biological systems. Proteases have been selected for discussion in those instances where natural substrates appear to have been identified. The studies reviewed point to the critical role that proteases play in protein processing and degradation.

Epithelial antibiotics induced at sites of inflammation.

The role of antimicrobial peptides in epithelial defense is not fully understood. An epithelial beta-defensin, lingual antimicrobial peptide (LAP), was isolated from bovine tongue and the corresponding complementary DNA cloned. LAP showed a broad spectrum of antibacterial and antifungal activities. LAP messenger RNA abundance was markedly increased in the epithelium surrounding naturally occurring tongue lesions. This increase coincided with the cellular hallmarks of acute and chronic inflammation in the underlying lamina propria, supporting a role for epithelial antimicrobial peptides as integral components of the inflammatory response.

Bone morphogenetic protein 2/4 in early fibromatous lesions of fibrodysplasia ossificans progressiva.

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder characterized by congenital malformation of the great toes and progressive heterotopic ossification in distinct anatomic patterns. Early preosseous lesions in FOP are clinically and histologically indistinguishable from the lesions of aggressive juvenile fibromatosis (AJF). Although the genetic defect in FOP is unknown, bone morphogenetic proteins (BMPs) 2 and 4 are plausible candidates genes. To determine if there is a difference in BMP 2/4 expression in the early fibromatous lesions of the two conditions, we performed immunohistochemical studies with a monoclonal antibody to BMP 2/4 on the earliest detectable fibromatous lesions of FOP and compared them with histologically identical lesions resected from children who had AJF. Fibromatous cells from the early FOP lesions exhibited immunostaining for BMP 2/4, whereas histologically indistinguishable fibromatous cells from AJF lesions showed no evidence of BMP 2/4 immunostaining. It is incumbent on all physicians who treat patients with suspected fibromatosis to examine the toes to rule out FOP and to avoid unnecessary diagnostic biopsies because surgical trauma induces further bone formation in patients who have FOP. However, if diagnostic confusion still exists and a biopsy is performed, immunostaining with BMP 2/4 antibody may resolve the diagnostic dilemma between FOP and AJF before the appearance of heterotopic ossification is observed in the FOP lesions. Our data suggest that the BMP 2/4 subfamily of secreted proteins may be involved in the pathogenesis of the FOP lesions.

In vitro susceptibility to pexiganan of bacteria isolated from infected diabetic foot ulcers.

During two clinical trials involving the treatment of 835 outpatients with infected diabetic foot ulcers, 2515 bacterial isolates, including 2337 aerobes and 178 anaerobes, were grown from cultures of the ulcers. The in vitro susceptibility of these isolates was determined to pexiganan, a peptide anti-infective evaluated in these clinical trials, and to other classes of antibiotics. Pexiganan demonstrated broad spectrum antimicrobial activity against Gram-positive and Gram-negative aerobes and anaerobes. The MIC90 values for the most common species among 1735 Gram-positive aerobes isolated, such as Staphylococcus aureus, coagulase-negative staphylococci, Group A streptococci, and Group B streptococci, were 16 micrograms/mL or less. Of 602 Gram-negative aerobes tested, the MIC90 values for pexiganan were 16 micrograms/mL or less for Acinetobacter, Pseudomonas, Stenotrophomonas, Citrobacter, Enterobacter, Escherichia, Klebsiella, and Flavobacterium species. Pexiganan had a MIC90 of 4 to 16 micrograms/mL against the anaerobic isolates of Bacteroides, Peptostreptococcus, Clostridium, and Prevotella species. Importantly, pexiganan did not exhibit cross-resistance with other commonly used antibiotics, including beta-lactams, quinolones, macrolides, and lincosamides. The broad spectrum in vitro antimicrobial activity of pexiganan against clinical isolates from infected diabetic foot ulcers supports its potential as a local therapy for infected diabetic foot ulcers.

Spinal deformity in patients who have fibrodysplasia ossificans progressiva.

We reviewed roentgenograms and clinical records in order to characterize the spinal deformity in forty patients who had an established diagnosis of fibrodysplasia ossificans progressiva. Twenty-six (65 per cent) of the patients had scoliosis, which, according to the clinical records and the recollection of the patients, had been present during childhood. Twenty-three (88 per cent) of the twenty-six curves were unbalanced c-shaped curves, while the remaining three (12 per cent) were balanced s-shaped curves. Twenty-one (91 per cent) of the twenty-three c-shaped curves involved the thoraco-lumbar or lumbar spine. The c-shaped curves ranged in magnitude from 15 to more than 80 degrees. Curves became rigid by early adulthood and many resulted in severe pelvic obliquity with impaired sitting or standing balance. An osseous bridge developed between the posterolateral aspect of the iliac crest and the posterolateral aspect of the rib cage in twenty-two (55 per cent) of the forty patients. Nineteen (86 per cent) of these twenty-two patients had scoliosis; there was a significant association between the development of scoliosis and the presence of the osseous bridge (p < 0.005). Ossification of the paravertebral muscles and fascia during the first decade of life limited the development of a normal thoracic kyphosis in ten (42 per cent) of twenty-four patients for whom lateral roentgenograms of the spine were available. A spinal orthosis was used to treat the scoliosis in two patients, but this method resulted in breakdown of the skin and failed to halt progression of the curve.(ABSTRACT TRUNCATED AT 250 WORDS)

Genetic transmission of fibrodysplasia ossificans progressiva. Report of a family.

Fibrodysplasia ossificans progressiva is a rare connective-tissue disorder characterized by congenital malformation of the great toes and by progressive heterotopic ossification of the tendons, ligaments, fasciae, and skeletal muscles. We document the genetic transmission of fibrodysplasia ossificans progressiva from a sporadically affected father to each of his three children: two daughters and a son. Previous consideration of a genetic etiology was based on the fact that the disease has been reported in several sets of monozygotic twins, that an increased paternal age has been associated with sporadic occurrences of the disorder, and that there have been several reports of genetic transmission in the remote past. Although an autosomal-dominant genetic transmission has long been suspected, the findings in the family reported on here provide confirmation for such inheritance and a basis for the diagnosis and counseling of patients who have this disease.

The histopathology of fibrodysplasia ossificans progressiva. An endochondral process.

In order to better characterize the biological features of fibrodysplasia ossificans progressiva, we reviewed the histopathological specimens from eleven patients (twelve biopsies) who had a confirmed diagnosis of the disease. All of the biopsies had been performed in children, to exclude the diagnosis of a malignant lesion. In no instance was the diagnosis of fibrodysplasia ossificans progressiva considered before the biopsy. The results of a lesional biopsy in all eleven patients revealed normal endochondral osteogenesis at heterotopic sites. The results of biopsy of an early lesion in six children were misinterpreted as revealing a diagnosis of fibromatosis or sarcoma before the roentgenographic appearance of ossification. Immunohistochemical studies of sections of the earliest lesion demonstrated S-100 antigen positivity before the histological appearance of differentiated osteochondral tissue. The presence of congenital malformation of the great toes and of postnatal heterotopic endochondral osteogenesis strongly suggests that fibrodysplasia ossificans progressiva is a disorder of defective induction of endochondral osteogenesis. This study established the predominant histopathological findings associated with fibrodysplasia ossificans progressiva and can serve as a basis for postulation of a candidate gene in the pathogenesis of the disorder. A lesional biopsy is not needed to make the diagnosis; biopsy uniformly exacerbates the condition and should be avoided.

Submandibular swelling in patients with fibrodysplasia ossificans progressiva.

Fibrodysplasia ossifcans progressive (FOP) is a rare genetic disorder characterized by congenital malformation of the great toes and by progressive heterotopic ossification of soft tissues. Although ankylosis of the temporomandibular joint occurs commonly in the late stages of the disease, only one well-documented case of submandibular heterotopic ossification in a patient who had FOP exists. Twelve (11 %) of our 107 patients who have FOP had submandibular heterotopic ossification that was mistaken initially in seven of the patients for mumps, angioneurotic edema, abscess, mononucleosis, or neoplasm. Two male patients and 40 female patients ranging in age from 6 to 47 years (mean, 21 years) were studied. Ten patients survived following assiduous precautionary measures. One patient who required emergency tracheostomy and ventilatory support also survived. Another patient died of inanition from chronic swallowing difficulty. An effective treatment program includes early identification of the submandibular flare-up, nutritional support, and glucocorticoid therapy. Submandibular swelling in patients who have FOP can be a medical emergency and requires intensive precautionary measures to avoid catastrophic clinical deterioration. These measures include avoidance of lesional manipulation, airway monitoring, and aspiration precautions. Submandibular swelling should be recognized as a variable feature of FOP.

Technetium-99m MDP demonstration of heterotopic ossification in fibrodysplasia ossificans progressiva.

The extensive heterotopic bone formation in patients with fibrodysplasia ossificans progressiva (FOP) has been documented previously with radiographs. A case in which a Tc-99m MDP bone scan showed increased uptake at sites well before ossification could be documented radiographically is described. This finding suggests that bone scans would likely be useful to monitor the extent of involvement with FOP and to detect areas of new activity.

Radiographic and scintigraphic features of modeling and remodeling in the heterotopic skeleton of patients who have fibrodysplasia ossificans progressiva.

To characterize the radiographic and scintigraphic features of modeling and remodeling in the heterotopic skeleton of patients who have fibrodysplasia ossificans progressiva, radiographs from 47 patients and radionuclide bone scans from 12 of those patients, all of whom had a confirmed diagnosis of the disease, were reviewed. A wide range of normal bone modeling and remodeling features was seen in the heterotopic skeleton of all but the youngest two (age, 1 year) of the 47 patients. Characteristic features of normal bone modeling identified on radiographs of the heterotopic skeleton included: (a) the development of tubular and flat bones with mature cortical and trabecular organization; (b) the presence of well defined cortical-endosteal borders enclosing medullary canals; and (c) the presence of metaphyseal funnelization in isolated ossicles or at sites of synostoses. Characteristic features of normal bone remodeling identified on radiographs of the heterotopic skeleton included: (a) the response of heterotopic bone to weight bearing stress with osteosclerosis of use and osteopenia of disuse, and (b) the resistance of heterotopic bone to fatigue failure with the absence of pathologic fractures and stress fractures. Radionuclide bone scans in 12 patients showed that remodeling of mature heterotopic bone occurred at a rate consistent with that of mature normotopic bone. This study documents the radiographic and scintigraphic features of a heterotopic skeletal system in 47 patients who have fibrodysplasia ossificans progressiva. These data provide additional support for the hypothesis that the genetic defect leading to the formation of a heterotopic skeleton involves normal skeletal morphogenesis at heterotopic sites.

Nucleosomal packaging of the thymidine kinase gene of herpes simplex virus transferred into mouse cells: an actively expressed single-copy gene.

We have studied the nucleoprotein structure of the herpes thymidine kinase gene introduced into mouse Ltk-aprt- cells by means of DNA-mediated gene transfer. Using the technique of Southern blotting, we examined staphylococcal digests of the nuclei from the relatively stable transformants that contain one or less integrated copies of the thymidine kinase gene per haploid genome. Out experiments show that, under selection for the active expression of this gene, it is packaged in nucleosomes with a repeat length identical to the average for the host mouse sequences.

Dense metaphyseal bands and growth arrest associated with isotretinoin therapy.

A 9-year-old boy, treated with high-dose isotretinoin therapy for fibrodysplasia ossificans progressiva, developed dense metaphyseal bands and growth arrest. Discontinuance of isotretinoin therapy was followed by gradual decrease of metaphyseal bands and resumption of clinical growth. The dense metaphyseal bands may be related to the known action of retinoids as modulators of chondrocyte phenotype and gene expression.

Fibrodysplasia ossificans progressiva: a clue from the fly?

Fibrodysplasia (myositis) ossificans progressiva (FOP) is a rare autosomal dominant disorder characterized by symmetrical congenital malformations of the blastemal anlage of the hands and feet and by progressive heterotopic chondrogenesis and ossification of the soft connective tissues. There is neither an established pathogenesis nor an effective treatment for this disabling disorder. We reevaluated the published data on the natural history of FOP and discovered an array of developmental gradients (characteristic patterns of disease expression) similar to developmental anomalies induced by pleiotropic mutations of the decapentaplegic (dpp) locus in Drosophila melanogaster. The protein encoded by the dpp locus is a member of the transforming growth factor-beta (TGF-beta) family of molecules. It shares 75% sequence homology with the c-terminal region of two recently cloned human bone morphogenetic proteins (BMP-2A, BMP-2B), also members of the TGF-beta family. The striking sequence identity across so large an evolutionary distance suggests that the BMP-2 genes in man and the dpp gene in the fly may be derived from a common ancestral gene. BMP is the only molecule discovered thus far that is capable of inducing endochondral ossification in vivo. Expression of endochondral bone formation is the basis for limb formation in embryogenesis, longitudinal bone growth in postnatal life, and local bone regeneration (fracture healing) following injury. We believe that FOP is a genetic disorder characterized by a disturbed developmental expression of this endochondral program and may represent a mutation resulting in a dominant gain of function. The developmental similarities between decapentaplegic in the fly and FOP in man suggest a useful model for the study of FOP. The use of such a model might be especially fruitful in suggesting a molecular basis for FOP.

Human and chick alpha 2(I) collagen mRNA: comparison of the 5' end in osteoblasts and fibroblasts.

Type I collagen, a heterotrimer of two alpha 1(I) chains and one alpha 2(I) chain, is the major structural protein of bone, skin, and tendon. The collagen of patients with bone diseases has been studied in skin fibroblasts instead of osteoblasts because the genes for type I collagen are single-copy genes. While these studies should detect structural changes in the gene, they might fail to detect defects in processes which are dependent on tissue-specific expression. The studies reported here sought to determine whether the expression of type I collagen in skin and bone was characterized by the use of alternate promoters or alternative splicing in the N-propeptide region. Primer extension and nuclease S1 protection experiments were used to analyze the structure of the alpha 2(I) mRNA from the 5' end of the gene through the N-telopeptide coding region (exons 1-6) in human and chick osteoblasts and fibroblasts. The protection and primer extension experiments using human and chick mRNA demonstrated identical routes of splicing in skin and bone at the first five splice junctions. These studies provide reassurance that information obtained from the study of type I collagen in fibroblasts may be extrapolated to bone.

A cross-species analysis of the cystic fibrosis transmembrane conductance regulator. Potential functional domains and regulatory sites.

To help elucidate the function of the cystic fibrosis transmembrane conductance regulator (CFTR), we have undertaken a cross-species analysis of the DNA sequence which encodes this protein. We have isolated and characterized the cDNA of the bovine homologue of CFTR. The deduced amino acid sequence shows high overall identity with the published sequences from human and mouse, although there is marked variability between the different potential functional domains. The region around human amino acid 508, which is deleted in 70% of cystic fibrosis chromosomes, is highly conserved across species; of the missense cystic fibrosis mutations reported to date, all of the amino acids in the normal human sequence are conserved in the bovine and mouse sequences. A single amino acid encoded by the human cDNA (Ser-434) is missing in the bovine sequence, and there are two amino acids encoded by the bovine sequence which are absent in the human. These all stem from in-frame 3-base omissions within the sequences. In addition to the cow, we amplified the DNA sequences encoding a portion of the R-domain from sheep, monkey, rabbit, and guinea pig. These sequences show relatively low overall sequence identity (63%), but nearly all of the potential protein kinase A and protein kinase C phosphorylation sites are conserved over all of the species examined. Our results suggest functional significance for certain highly conserved residues and putative domains within CFTR.

Fibrodysplasia ossificans progressiva: CT appearance.

Twelve patients with fibrodysplasia ossificans progressiva were studied with computed tomography (CT). Characteristic swelling of the muscular fascial planes could be identified on CT scans prior to the development of ectopic ossification. Ossification could be seen on CT scans before it was apparent on plain radiographs. The pattern of ossification was similar to that seen at pathologic study, with multifocal sites developing adjacent to and extending around muscles. The appearance on CT scans confirms the hypothesis that the initial focus in fibrodysplasia ossificans progressiva is in the connective tissue.

Failure of surgery and isotretinoin to relieve jaw immobilization in fibrodysplasia ossificans progressiva: report of two cases.

Two patients with fibrodysplasia ossificans progressiva who presented with jaw immobilization due to formation of bone between the maxilla and mandible were treated with surgical resection of their ectopic bone in conjunction with experimental, adjunctive medical therapy using isotretinoin. Both patients had recurrence of their ectopic ossification within 2 months of surgery. Surgery to remove joint-bridging ossifications in FOP is not recommended.

Permanent heterotopic ossification at the injection site after diphtheria-tetanus-pertussis immunizations in children who have fibrodysplasia ossificans progressiva.

In patients who have fibrodysplasia ossificans progressiva, routine childhood diphtheria-tetanus-pertussis immunizations administered by intramuscular injection caused a significant risk of permanent heterotopic ossification at the site of injection (p < 10(-8)), whereas measles-mumps-rubella immunizations administered by subcutaneous injection posed no significant risk. Intramuscular injections should be avoided, if possible, once a diagnosis of fibrodysplasia ossificans progressiva has been established.