Osteoradionecrosis, an increasing indication for microvascular head and neck reconstruction.

Better cancer treatment has led to a steadily growing population of cancer survivors suffering from late adverse effects after cancer treatment. The aim of this study was to investigate whether there has been an increase in free flap reconstruction due to osteoradionecrosis (ORN). A retrospective review was conducted to identify all consecutive head and neck free flap reconstructions performed over an 18-year period (1995-2012) at Karolinska University Hospital. A total of 235 free flaps were identified. Cases were divided into two groups: head and neck cancer reconstructions and ORN reconstructions. A comparison between the two groups showed longer survival (P<0.001) and higher rates of late complications (P<0.001) among ORN cases. ORN as an indication for reconstruction increased over time, from 7.0% of the total number of free flaps performed in 1995-2000, to 15.2% during the period 2001-2006, and to 27.3% in 2007-2012 (P<0.001). This, in accordance with the results of other studies, highlights the importance of the appropriate allocation of resources within the healthcare system to treat this patient group within the steadily increasing population of cancer survivors.

Antineutrophil Cytoplasmic Antibodies in Inflammatory Bowel Disease and Collagenous Colitis: No Association with Lactoferrin, ß-Glucuronidase, Myeloperoxidase, or Proteinase 3.

: Perinuclear antineutrophil cytoplasmic antibodies (P-ANCA) occur frequently in ulcerative colitis (UC) but not in Crohn's disease (CD). Their pathogenetic importance is unknown, and studies of associated antigens have been inconsistent. Indirect immunofluorescence technique was used to screen the occurrence of antineutrophil cytoplasmic antibodies in 36 patients with UC, in 37 patients with CD, in 38 patients with collagenous colitis (CC), and in 190 controls. Enzyme immunoassays (EIAs) were used to detect the target antigen(s) by using lactoferrin (Lf), ß-glucuronidase (ß-Glc), myeloperoxidase (MPO), and proteinase 3 (PR3) as the substrates. P-ANCA was found in 23 (63.9%) of 36 of the patients with UC, in two (5.4%) of 37 with CD, in four (10.5%) of 38 with CC, and in four (2.1%) of 190 of healthy controls. No case of cytoplasmic staining pattern (C-ANCA) was found. With EIA, P-ANCA in IBD or CC was not found to be associated with reactivity to Lf, ß-Glc, MPO, or PR3, which confirms findings reported by others. P-ANCA was found in a higher frequency in UC than in CD or CC. The antigens of P-ANCA remain unidentified.

Bacterial adherence to endothelial cells from rat heart, with special regard to alpha-hemolytic streptococci.

Endothelial cells were prepared from rat heart, and the ability of various bacterial species to adhere to these cells was investigated, with special regard to alpha-hemolytic streptococci. The endothelial cells were identified by morphology and by the presence of F VIII antigen. Seventeen bacterial strains representing various species were used in the in vitro adherence tests; 10 strains represented various strains of alpha-hemolytic streptococci, 5 of which were isolated from patients with infective endocarditis and 5 from the throats of healthy individuals; 2 were Neisseria meningitidis, 2 N. gonorrhoeae, 2 S. aureus and 1 E. coli. The highest adherence rate was found for alpha-hemolytic streptococci isolated from patients with infective endocarditis. The difference compared to carrier strains of alpha-hemolytic streptococci and to the other tested strains was statistically highly significant.

Increased adherence to vaginal epithelial cells and phagocytic killing of gonococci and urogenital meningococci associated with heat modifiable proteins.

Urogenital Neisseria meningitidis were characterized with regard to serogroup, colony morphology, the presence of heat modifiable proteins (HMP), attachment to human vaginal and buccal epithelial cells, and phagocytic killing by polymorphonuclear leukocytes. The findings were compared with those on gonococci, and with those on meningococci isolated from blood or cerebrospinal fluid, with regard to colony morphology, HMP and piliation. The opacity colony morphology characteristic could be used to predict the presence of HMP in gonococci but not in meningococci, and sodium dodecyl sulphate polyacrylamide gel electrophoresis had to be used to demonstrate this surface protein. The urogenital meningococci, serogroup Y, attached significantly more efficinetly to vaginal epithelial cells in the presence of HMP and behaved in this respect like those of gonococci. Gonococci and meningococci containing HMP were more sensitive to phagocytic killing than those without HMP. Meningococci from opaque and transparent colonies and isolated from patients with meningococcal disease had no demonstrable HMP. They showed low adherence to vaginal and buccal epithelial cells, with no difference between organisms from opaque or transparent colonies.

A reference procedure to study chemiluminescence induced in polymorphonuclear leukocytes by Neisseria meningitidis.

Luminol-enhanced chemiluminescence (CL) was used to study the ability of various strains of Neisseria meningitidis (MC) to induce oxidative metabolism of polymorphonuclear leukocytes (PMNL); an indirect measure of phagocytic activity. To circumvent variations related to different PMNL donors, a MC serogroup X strain was used as a control for indexing the CL responses induced by other MC strains. This procedure, with pooled serum from healthy blood donors to standardize opsonising conditions, gave reproducible and comparable results, irrespective of PMNL donors. Under these conditions, there was a highly significant difference between pathogenic and non-pathogenic MC strains as regards their ability to induce CL responses (p less than 0.001). The results indicated that the differences were due partly to opsonizing antibodies, partly to other differences related to pathogenicity of tested MC strains. These differences in leukocyte/MC interaction were also confirmed by phagocytic-killing experiments. The index procedure of CL measurements may be a suitable method to study the appearance of natural immunity to MC disease, as well as the pathogenicity of particular MC strains.

Mobiluncus species in bacterial vaginosis: aspects of pathogenesis.

Mobiluncus is an anaerobic motile rod associated with bacterial vaginosis. In this work, luminol-enhanced chemiluminescence was used to study the ability of Mobiluncus spp. from the vaginas of women with bacterial vaginosis to induce, in the presence of normal adult serum, oxidative metabolism of polymorphonuclear leukocytes, which is an indirect measure of phagocytic activity. M. curtisii induced a significantly (p less than 0.05) lower response than M. mulieris, which indicates that M. curtisii escapes phagocytosis more easily. Indirect immunofluorescence assays showed IgG antibodies to M. curtisii at significantly (p less than 0.01) higher titres than to M. mulieris in women with bacterial vaginosis. The titres were higher in patients with bacterial vaginosis than in women without vaginosis and healthy men. No antibodies to Mobiluncus spp. of secretory IgA type were found in vaginal washings. These results indicate that M. curtisii is a more virulent species than M. mulieris, and agree with reports of M. curtisii found in postoperative and extragenital infections.

Early development in healthy children of serum opsonins against nonpathogenic Neisseria meningitidis.

In an earlier study, with the use of chemiluminescence (CL) and phagocytic killing, we could show that in the presence of serum from healthy adults polymorphonuclear leukocytes (PMNL) efficiently handle nonpathogenic Neisseria meningitidis strains, in sharp contrast to those associated with clinical disease. The major part of this difference was dependent on serum factors. In the present study 84 serum samples from children 1-3, 4-6, 7-9, and 10-14 years old were studied by the CL technique according to their ability to opsonize meningococci. There was a highly significant difference (p less than 0.001) in all four age groups when the CL indexes obtained with the pathogenic meningococci of the serogroups A, B and C were compared with those of the nonpathogenic menigococci: serogroup 29E and nongroupable meningococci. These findings imply that the ability to opsonize so-called nonpathogenic meningococci is developed early in life and may explain why they are only occasionally able to cause disease.

Detection of antibody responses in rabbits hyperimmunized with Campylobacter pylori. Enzyme immunoassay indicates extensive antigenic similarities.

Acid glycine extracts from four Campylobacter pylori strains and one GCLO strain were used as antigens in enzyme immunoassay (EIA). Immune responses of rabbits immunized with C. pylori strains, the GCLO-strain and other campylobacter strains were studied. All 14 rabbit antisera against C. pylori reacted with all four C. pylori extracts and there were extensive cross-reactions between these extracts. Antisera against C. jejuni and C. coli strains did not react with a C. pylori extract but reached with the GCLO antigen. Acid glycine extracts proved to be very satisfactory in C. pylori serology and the use of an extract prepared from only one strain instead of a combination of strains seems justified.

Experimentally induced ileal ulcers in rats. Formula diet is a preventive factor.

We recently described an experimental model in the rat to create recurring chronic ileal inflammation including ulceration. This model is dependent on an "in vivo culture" of normal intestinal contents. In the present experimental study we examined the effect of a polymeric and a hydrolyzed formula diet on the formation of ulcerating lesions using our rat model. Two groups of rats (twenty in each group) were fed either one of these formula diets eight weeks prior to the experimental procedure and this diet was continued until sacrifice eight weeks later. Twenty control rats also underwent the experimental procedure but were fed standard rat pellets for the same time periods. At sacrifice 60% of the control rats had developed ileal ulcers. None of the rats fed the formula diets developed macroscopic ileal inflammation or ulceration. The effectiveness of formula diets in inducing remission in Crohn's disease in humans may be linked to alterations in the intestinal microflora. We hypothesize that the formula diets in this experiment exerted a protective effect against ileal ulceration by altering the ileal microflora. Preliminary studies support this hypothesis but need to be expanded.

Formula diet is a preventive factor in experimentally induced ideal ulcers in rats. A bacteriological study.

We earlier described an experimental model to create recurring chronic ileal inflammation with ulceration in the rat. A 2 cm segment of the distal ileum is excised but left attached to its intact mesentery; the ileum is reanastomosed. The ileal segment will seal off its open ends and a cyst-like structure of varying size will be formed, containing mucus, cell debris and bacteria. Approximately two thirds of the animals develop chronic inflammation with ulceration proximal to the ileal anastomosis. The ileal cyst and the surgical procedure on the distal ileum were shown to be prerequisites of the rat model for the development of lesions. We recently described that, in contrast to rats fed a standard diet, rats fed a hydrolyzed formula diet never developed inflammation or ulceration when subjected to the experimental procedure. In the present study we confirmed these observations and showed that the normal ileal flora (NIF) and the ileal cyst flora (ICF) were significantly influenced by the diets. The bacterial counts of both the aerobic and anaerobic NIF were 2 10log lower, i.e. > or = 99%, in rat fed the formula diet as compared to in those fed standard rat pellets. The NIF of the former group was represented by more aerobic species than the NIF of rats on the standard diet. Compared to the NIF there was a parallel increase in the bacterial counts of the ICF by approximately 2 10log CFU values in both groups of rats. The mean number of anaerobic species, mainly Gram-negative rods of the ICF, increased by approximately 70% in the rats on the standard diet that developed ileal ulceration, whereas identified aerobic species of the ICF decreased by 61% in rats on the formula diet and by 46% in those on the standard diet that did not develop ileal ulceration. The number of anaerobes in those groups of rats remained unchanged. The significant bacteriological differences between the rats that developed ileal ulcers and those which did not indicate that bacteria may be involved, directly or indirectly, in the development of chronic ileal ulceration.

Rapid serotyping of groups A, B, and C meningococci by rocket-line immunoelectrophoresis and co-agglutination.

Rocket-line immunoelectrophoresis (R-LIE) with antigen containing intermediate gel, and co-agglutination utilising protein A-containing staphylococci coated with specific antibodies, were adapted for serotyping the prototypes of group B meningococci. Both were found to have the same specificity as agar gel double diffusion (AGDD) but they were more sensitive and more rapid than AGDD. R-LIE required, like AGDD, the extraction of relatively large quantities of bacteria, while the co-agglutination method, performed as a slide agglutination tests with results within a few minutes and no need of special equipment, required only a small amount of heated whole meningococcal cells. Meningococcal strains of serogroups B, C, and A from patients with carriers were serotyped and the results with all three methods were in agreement.

Diagnosis of urogenital gonorrhoea by detecting gonococcal antigen with a solid phase enzyme immunoassay (Gonozyme).

A solid phase enzyme immunoassay (Gonozyme) was used to demonstrate gonococcal antigen in urogenital specimens. Urethral specimens from 101 men and cervical specimens from 150 women were examined, and the diagnostic yields were compared with those obtained by culture. The Gonozyme test was positive in 25 patients, 15 men and 10 women, and negative in 226 patients. Gonococci were isolated by culture in 23 of the patients, 12 men and 11 women. The Gonozyme test gave false-negative results in two men and one woman patient. The sensitivity of the test was 87% for the men and 91% for the women. Correspondingly, the test specificity was 94.3% for the men and 100% for the women, the predictive value of positive test 80% and 100%, and that of negative test 97.7 and 99.3% respectively. The Gonozyme test does not allow antibiotic sensitivity testings but has the advantage of rapidity and is not dependent upon viable organisms. The test is an attractive alternative to culture procedures for screening women patients with symptomatic or asymptomatic gonorrhoea.

Ultrastructural study of two patterns in the interaction of Helicobacter pylori with neutrophils.

As evidenced by electron microscopy, unopsonised bacteria of some Helicobacter pylori strains were readily taken into cytoplasmic vacuoles of human neutrophils; those of other strains were only rarely absorbed. The strains engulfed like this also induced strong oxidative burst reactions in neutrophils, as measured by chemiluminescence. This has been associated with peptic ulcer disease, in the past. The ultrastructural interaction patterns agreed with the reaction patterns shown by chemiluminescence.

Culture diagnosis of meningococcal carriers: yield from different sites and influence of storage in transport medium.

Different specimens and techniques have been used in the diagnosis of carriers of Neisseria meningitidis, reflecting the uncertainty about the optimal diagnostic procedure. In the present investigation the culture yield of meningococci from throat specimens was compared to that from nasopharyngeal speimens in 178 persons: 44 carriers were diagnosed. All of them were detected by culture of throat specimens while 34% of them would have remained undiagnosed if only nasopharyngeal specimens had been examined. Storage of throat specimens in a transport medium for 24 hours before culture gave a negative culture for meningococci in 41% of the carriers. This loss was surprisingly high, the reasons for which are discussed.

Co-expression in Helicobacter pylori of cagA and non-opsonic neutrophil activation enhances the association with peptic ulcer disease.

AIMS: To investigate the association of cagA positivity and non-opsonic neutrophil activation capacity in wild-type Helicobacter pylori strains with peptic ulcer disease or chronic gastritis only. METHODS: Helicobacter pylori were isolated from antral biopsies of 53 consecutive patients with chronic antral gastritis, of whom 24 had peptic ulcer disease endoscopically. The presence of cagA, a marker for the cag pathogenicity island, was determined by polymerase chain reaction with specific oligonucleotide primers, and non-opsonic neutrophil activation capacity by luminol enhanced chemiluminescence. RESULTS: The cagA gene was present in 39 of 53 (73.6%) strains, 20 of which (83.3%) were from the 24 patients with peptic ulcer disease and 19 (65.5%) from the 29 patients with chronic gastritis only. Non-opsonic neutrophil activation was found in 29 (54.7%) strains, 16 of which (66.7%) were from patients with peptic ulcer disease, and 13 (44.8%) from those with chronic gastritis. Non-opsonic neutrophil activation was found more frequently in cagA+ than cagA- strains (59% v 42.9%). Whereas four of the 14 cagA- strains and eight of the 24 non-opsonic neutrophil activation negative strains were from patients with peptic ulcer disease, only two of 24 (8.3%) peptic ulcer disease strains expressed neither cagA nor non-opsonic neutrophil activation. The cagA gene and non-opsonic neutrophil activation capacity were co-expressed in 14 of 24 (58.3%) strains from patients with peptic ulcer disease, and in nine of 29 (31%) strains from individuals with chronic gastritis. CONCLUSIONS: Positivity for cagA and non-opsonic neutrophil activation occur independently in wild-type H pylori strains. However, co-expression of the two markers enhanced the prediction of peptic ulcer disease.

Diagnosis of Neisseria infections by defined immunofluorescence. Methodologic aspects and applications.

Defined IF techniques have been used in our laboratories for the past 15 years for diagnosing meningococcal and gonococcal infections. Conjugates against these bacteria give desired specific staining (DSS) but also undersired specific staining (USS) due to cross-reacting antigens. USS is controlled by absorption. Nonspecific staining (NSS) of protein A-containing S. aureus caused by the reaction with the Fc part of the IgG molecule is blocked by adding RB 200-labeled antiS. aureus globulins to diluted FITC conjugates against gonococci or menigococci. Nss is also avoided by using FITC-labeled F(ab')2 fragments of IgG. By optimal labeling and separation of unlabeled FITC and heavily labeled molecules on Sephadex G-25 at low ionic strength and low pH, nonspecific reactions with tissues are reduced to a minimum. Defined IF can be utilized for diagnosing pathogenic Neisseria by examination of smears prepared from clinical specimens, as confirmative tests of oxidase-positive colonies, and for demonstrating these bacteria in tissue efflorescences and tissue biopsies. It is stressed that this technique should always be used in conjunction with conventional methods.

Studies on rabbit hyperimmune, patient and blood donor serum with regard to bactericidal activity and serum antibodies against anaerobic curved rods from patients with bacterial vaginosis.

Serum specimens from patients with bacterial vaginosis (BV) from whom anaerobic curved rods (ACR) were isolated, from healthy female and male blood donors and from rabbits immunized with ACR were tested for anti-ACR antibodies by indirect immunofluorescence and for bactericidal activity. The rabbit sera had antibody titres ranging from 1 : 160 to 1 : 2560 but no bactericidal activity could be demonstrated. Very low titres (1:10) of IgM antibodies were detected in serum from a few patients as well as from a few female and male blood donors, whereas no IgG or IgA antibody titres (less than 1:10) were demonstrated. No bactericidal activity was found in the human sera against ACR. We conclude that no systemic antibody response can be demonstrated in patients with BV from whom such organisms are isolated. Moreover, these organisms behave differently from other Gram-negative bacterial species, in that they are resistant to normal serum as well as the specific antibody-complement-mediated bactericidal activity.

An in vitro study of the attachment to vaginal epithelial cells of anaerobic curved rods, Bacteroides bivius and Bacteroides disiens.

An in vitro test system was used to study the influence of different pH levels on the adherence of anaerobic curved rods (ACR) of the long (LCR) and short type (SCR), of Bacteroides bivius and B. disiens, to vaginal epithelial cells (VEC) obtained from healthy women. There was a significant increase in the adherence of ACR (p less than 0.05) and B. bivius (p less than 0.05) at decreasing acidity. LCR adhered significantly better than SCR at pH 7.5 as compared with pH 4.0 (p less than 0.01). 'Clue cells' seen in wet mounts from vaginal discharge of women with bacterial vaginosis were very similar to VEC covered with ACR in in vitro tests.

A serological study of anaerobic curved rods with rabbit hyperimmune antisera.

Anaerobic curved rods (ACR) isolated from vaginal discharge of women with a diagnosis of bacterial vaginosis (BV) were investigated with regard to bacteriologic and serologic characteristics. According to morphologic criteria, long curved rods (LCR) which were sensitive to metronidazole, and short curved rods (SCR) which showed resistance to metronidazole, were recognized. LCR degraded several sugars, whereas SCR degraded only a few carbohydrates. With the use of hyperimmune polyclonal rabbit anti-ACR antibodies, heat-labile and heat-stable antigens were demonstrated in whole cells of both LCR and SCR strains by co-agglutination (CoA). Heat-labile antigens which could be flagellae or other surface components seemed to be responsible for inter-group and inter-strain specificity and did not cross-react between LCR and SCR. Two types of heat-stable antigens, one resistant to 120 degrees C, and one resistant to 100 degrees C but not 120 degrees C, were demonstrated in both LCR and SCR. These heat-stable antigens showed considerable one-way and two-way cross-reactivities within the LCR group, and one-way cross-reactivities were also demonstrated between LCR and SCR strains. These cross-reactivities were also demonstrated by indirect immunofluorescence (IFL) in untreated whole cells. The results show that IFL could be used to identify untreated whole cells and CoA to identify heated bacteria.

Heterogeneity of Campylobacter pylori as demonstrated by co-agglutination testing with rabbit antibodies.

The indirect immunofluorescence (IFL) and the co-agglutination (CoA) methods were used to study the serology of Campylobacter pylori strains isolated from patients in different countries (Sweden, Finland, Canada and Australia). Antisera were obtained from rabbits immunized with whole cell antigens. With IFL tests the highest serum titers were obtained with C. pylori strains and their homologous antisera. These tests also showed that all the tested strains contained cross-reactive antigens. With the use of the CoA technique strain or type specific heat labile and heat stable antigens were demonstrated, and a provisional "seropattern" of a particular strain could be defined with selected CoA reagents. With the use of such reagents we were able to show that a patient may be infected with multiple C. pylori strains with different sets of surface antigens. The clinical and epidemiological implications of this serological heterogeneity of C. pylori are discussed.