Infantile systemic hyalinosis in siblings: clinical report, biochemical and ultrastructural findings, and review of the literature.

A boy presented at age 3.5 months with joint contractures, restlessness, and pain on handling. His skin was thickened and there were livid-red macular lesions over bony prominences. Infantile systemic hyalinosis (ISH) was diagnosed, a presumably autosomal recessive, progressive, and painful disorder of as yet unknown pathogenesis. Observation over three years confirmed the diagnosis as typical changes, such as nodules on both ears, pearly papules in the perinasal folds and on the neck, fleshy nodules in the perianal region, and gingival hypertrophy, developed. Skin lesions and painful joint contractures progressed in spite of intense physiotherapy, and at age 3, the child had marked motor disability. The central nervous system (CNS) appeared to be intact and the infant showed normal mental development. Radiologic findings included marked generalized osteopenia, osteolytic erosions in the metaphyses of the long bones, and cortical thinning. Electron microscopy of two skin biopsies demonstrated deposition of floccular amorphous substance that was abundant around, and appeared to originate from, small blood vessels in the dermis, partially interfering with collagen fiber formation. Lysosomal inclusions were not seen. Serum acid hyaluronidase activity was within the normal range, and the synthesis of hyaluronic acid and proteoglycans in cultured skin fibroblasts was similar to that of control cells. A younger sister presented at age two months with painful joint contractures and discrete livid-red macules over both malleoli, and showed a similar progression of the disorder over the first year of life. The diagnosis of ISH should be considered in infants and children presenting with painful joint contractures and skin lesions. The pathogenesis of this disabling and disfiguring disorder remains unclear. Our data confirm probable autosomal recessive inheritance, and do not support lysosomal storage, hyaluronidase deficiency, or a primary collagen disorder, but indicate that the amorphous material accumulating in the skin and articular soft tissues may originate from the blood circulation.

Temporal and local appearance of alkaline phosphatase activity in early stages of guided bone regeneration. A descriptive histochemical study in humans.

Alkaline phosphatase (ALP) catalyzes the hydrolysis of phosphate esters and it seems to be a prerequisite for normal skeletal mineralization. Also, ALP is the most widely recognized marker of osteoblast phenotypes. By a tissue regenerative technique called Guided Bone Regeneration (GBR), it is possible nowadays to regenerate small bony defects. The aim of the present study was to investigate early events in bone healing and neogenesis by studying histochemically the temporal and local appearance of the marker Alkaline Phosphatase (ALP) in a GBR model system. Nine healthy volunteers (5 males, 4 females, mean age 31.7 years) participated in the experiment. After raising a mucoperiosteal flap from the mandibular second molar to the retromolar area in each volunteer, a hollow titanium test cylinder was placed into a congruent bony bed and the coronal end of the cylinder was closed with an ePTFE-membrane. Then the flap was adapted and sutured to obtain primary wound closure. After 2, 7 and 12 weeks, the regenerated tissue within the cylinders was harvested. Histologically, ALP activity was observed associated with the osteoid seams in the very basal part of the regenerate where new bone trabeculae were in the process of being formed. More coronally, large round cells seemed to secrete an ALP-positive substance since in the center of such cell clusters strong ALP activity located extracellularly was detected. In the present experiment, ALP seemed to have been an early sign of osteoblast secretion of a matrix which subsequently was determined to become osteoid. ALP activity was never seen isolated within connective tissue and away from bone. This is an indication that its source is linked to existing bone. The present study has documented for the first time the appearance of ALP activity in guided bone regenerations in humans. It has revealed that: 1) Osteogenesis in guided bone regeneration is preceded by localized, marked expression of ALP in an organized connective tissue environment. 2) Bone neogenesis is an early event in this experimental setup and may be detected already 2 weeks after wounding. 3) Expression of ALP and subsequent bone neogenesis is originating from and topographically linked to pre-existing bone structures.

Identification and characterization of an immunogenic outer membrane protein of Campylobacter jejuni.

We cloned and expressed in Escherichia coli a gene encoding an 18-kDa outer membrane protein (Omp18) from Campylobacter jejuni ATCC 29428. The nucleotide sequence of the gene encoding Omp18 was determined, and an open reading frame of 165 amino acids was revealed. The amino acid sequence had the typical features of a leader sequence and a signal peptidase II cleavage site at the N-terminal part of Omp18. Moreover, the sequence had a high degree of similarity to the peptidoglycan-associated outer membrane lipoprotein P6 of Haemophilus influenzae and the peptidoglycan-associated lipoprotein PAL of E. coli. Southern blot analysis in which the cloned gene was used as a probe revealed genes similar to that encoding Omp18 in all species of the thermophilic group of campylobacters as well as Campylobacter sputorum. All campylobacters tested expressed a protein with a molecular mass identical to that of Omp18. The protein reacted immunologically with polyclonal antibodies directed against Omp18 from C. jejuni. PCR amplification of the gene encoding Omp18 with specific primers and subsequent restriction enzyme analysis of the amplified DNA fragments showed that the gene for Omp18 is highly conserved in C. jejuni strains isolated from humans, dogs, cats, calves, and chickens but is different in other Campylobacter species. In order to obtain pure recombinant Omp18 protein for serological assays, the cloned gene for Omp18 was genetically modified by replacing the signal sequence with a DNA segment encoding six adjacent histidine residues. Expression of this construct in E. coli allowed purification of the modified protein (Omp18-6xHis) by metal chelation chromatography. Sera from patients with past C. jejuni infection reacted positively with Omp18-6xHis, while sera from healthy blood donors showed no reaction with this antigen. Omp18, which is an outer membrane protein belonging to the family of PALs is well conserved in C. jejuni and is highly immunogenic. It is therefore a good candidate as an antigen for the serological diagnosis of past C. jejuni infections.

Identification of Campylobacter jejuni on the basis of a species-specific gene that encodes a membrane protein.

To facilitate discrimination between the closely related enteropathogens Campylobacter jejuni and C. coli, unique differences in antigenic surface structure were examined. A genomic library of C. jejuni 81116 was constructed in plasmid pBluescriptIISK- and expressed in Escherichia coli K-12. Rabbit hyperimmune serum raised against C. jejuni ATCC 29428 recognized a clone expressing a C. jejuni 24-kDa membrane-associated protein. Antiserum raised against sonicated recombinant E. coli expressing the 24-kDa protein reacted with C. jejuni, whereas C. coli did not react specifically. Determination of the nucleotide sequence of the DNA insert of this recombinant plasmid revealed an open reading frame encoding 214 amino acids; the gene was designated mapA; and its gene product was designated MAPA. The 18 N-terminal amino acid residues constitute a signal sequence characteristic of prokaryotic membrane lipoproteins. In a dot blot hybridization assay with a mapA probe, 120 clinical isolates of C. jejuni were unequivocally discriminated from 126 other campylobacters, including 34 C. coli isolates. A PCR test based on the mapA sequence was developed for identification of C. jejuni. A PCR product was obtained with all of the clinical isolates of C. jejuni tested from human, dog, cat, bovine calf, and chicken sources. Recombinant MAPA with an added C-terminal six-histidine tail was affinity purified and used to immunize rabbits. The rabbit anti-MAPA serum specifically recognized the protein in whole cells of C. jejuni on Western blots (immunoblots). The MAPA protein was present in all of the C. jejuni strains tested and was absent in C. coli and related campylobacters.

Eimeria tenella: characterization of a 5S ribosomal RNA repeat unit and its use as a species-specific probe.

The oocysts of Eimeria tenella, one of the most pathogenic of several species causing chicken coccidiosis, are difficult to distinguish microscopically from several other infective Eimeria species. One copy of a gene coding for 5S ribosomal RNA has been cloned from E. tenella and sequenced. A coding region of 120 nucleotides and an intergenic region of 608 nucleotides together make up a 5S rRNA repeat unit, of which there are many copies tandemly repeated in the genome. The intergenic region is species-specific and sequences derived from it can be used for species identification by polymerase chain reaction (PCR). The PCR procedure described here is particularly sensitive, with fewer than 10 oocysts sufficing to give a positive result. This method may also be useful for the identification by PCR of other cyst-forming parasites.

Analysis of hemolysin operons in Actinobacillus pleuropneumoniae.

Among the twelve different serotypes of Actinobacillus pleuropneumoniae, the causative agent of swine pleuropneumonia, a strongly active hemolysin I (HlyI) is produced by serotypes which are particularly virulent, and less active hemolysin II (HlyII) is produced by all serotypes except type 10. In the serotypes 1, 5a, 5b, 9, 10 and 11, which produce HlyI, the hemolysin (hly) operon consists of a structural hlyIA gene, encoding pre-HlyI, an activator gene, hlyIC, necessary for the activation of pre-Hly to active Hly, and two genes, hlyIB and hlyID, involved in Hly secretion. These genes are clustered in the order, hlyICABD. This is characteristic to RTX toxin (repeats in the structural toxin) operons. The HlyII operons in all serotypes producing HlyII consist only of the pre-HlyII-encoding gene, appA, and its activator gene, appC. The serotypes, which produce HlyII, but not HlyI, contain a truncated HlyI operon, with the promoter, hlyIB and hlyID, and a small segment of the C terminus of hlyIA. This partial HlyI operon might have been formed by deletion of hlyIC and most of hlyIA. In serotype 3, which produces HlyII, but no HlyI, and which releases only minute amounts of this Hly into the growth medium, none of the hlyI genes and consequently no Hly secretion genes were found. The above results postulate that HlyII is secreted via the products of hlyIB and hlyID, and explain the low amount of HlyII secreted by serotype 3. Cloning and analysis of the structural genes encoding pre-HlyI and pre-HlyII among the different serotypes revealed differences in the hlyIA genes which are highly similar in the serologically related serotypes 1, 9 and 11, and differ from the serotypes, 5a, 5b and 10. The hlyIIA genes, in contrast, seem to be conserved in all serotypes.

[10 years' experience using a modified Shouldice surgical technic for inguinal hernia in adults. II. Which factors modify the recurrence of inguinal hernia?]. / 10 Jahre Erfahrung mit einer modifizierten Operationstechnik nach Shouldice für Inguinalhernien bei Erwachsenen. II. Welche Faktoren beeinflussen die Rezidivgenese von Inguinalhernien?

Many factors seem to influence the recurrence rate after adult inguinal hernia repair. A statistical analysis of data derived from 726 transversalis fascia repairs examined by the authors (with a follow-up rate of 82.5% and a mean follow-up time of 5.5 years) revealed a significantly higher recurrence rate in patients with chronic bronchitis (p less than 0.05) or with postoperative complications (p less than 0.001). Lower recurrence rates were found after resection of lipomas of the cord (p less than 0.01) or cremasteric muscle resection (p less than 0.05). No significant difference of recurrence rate could be established for following parameters: Sex, side, age distribution, profession, prostatism, obesity, type of hernia (direct, indirect, combined, sliding), suture material (silk, polyglycolic acid), surgeon, anesthesia (local, spinal, full), elective or emergency operation, and whether the repair was unilateral or simultaneously bilateral. Recurrent repairs showed no significantly higher recurrence rate than primary repairs.

[10 years' experience using a modified Shouldice surgical technic for inguinal hernia in adults. I. Method and results in 726 operations with follow-up]. / 10 Jahre Erfahrung mit einer modifizierten Operationstechnik nach Shouldice für Inguinalhernien bei Erwachsenen. I. Methode und Resultate bei 726 nachkontrollierten Operationen.

Since 1973 we have used a modified Shouldice repair to treat inguinal hernias in adults. The repair was performed on unselected patients. The procedure was performed by both senior surgeons and surgeons in training. Over a 10-years period, 880 transversalis fascia repairs were done and 726 (82.5%) were examined by the authors 12-142 months after the operation (mean time 66.1 months). In 94.2% of the 726 repairs the patients were satisfied with the result. Follow-up studies revealed testicular atrophy in 0.6%, deep wound infection in 0.8% and an overall recurrence rate of 5.8%. The recurrence rate was not significantly different after direct, indirect, combined or sliding hernias. In 25% of recurrences the patients were asymptomatic and unaware of the recurrence.