bluuebunny/crossref_metadata_2025 huggingface dataset:
First branchial cleft cysts: Clinical update
Authors: Finn Douglas G. , Buchalter Ira H. , Sarti Edward , Romo Thomas , Chodosh Paul
Published: September 2006

First branchial cleft cysts develop as a result of incomplete fusion of the cleft between the first and second branchial arches and give rise to two distinct anomalies, termed type I and type II anomalies. Type I anomalies are purely ectodermal while type II anomalies exhibit ectodermal and mesodermal elements. The type II anomaly incorporates some portion of the first and second arch as well as the cleft.

Type I lesions are extremely rare. They appear histologically as cysts lined by squamous epithelium. Clinically, they present as a cystic mass or fistula posterior to the pinna and concha. The cyst is usually superior to the main trunk of the facial nerve and ends in a cul‐de‐sac on or near a bony plate at the level of the mesotympanum.

Type II lesions are more numerous and represent a duplication of both membranous and cartilaginous portions of the external auditory canal. They contain skin as well as adnexal structures and cartilage and may be associated with the parotid gland. Most frequently they are associated with fistulae in the concha or external auditory canal as well as fistulous openings in the neck.
Prostaglandins, leukotrienes, and other arachidonic acid metabolites in nasal polyps and nasal mucosa
Authors: Jung Timothy T. K. , Stewart Robert , Juhn S. K. , Hwang Daniel
Published: September 2006

Prostaglandins (PGs) and leukotrienes (LTs) are known to play an important role in allergic inflammatory reactions. The triad of aspirin sensitivity, nasal polyposis, and asthma led us to suspect that PGs, LTs and other arachidonic acid metabolites may be involved in the pathogenesis of nasal polyps. The purpose of this study was to determine arachidonic acid metabolites and to measure concentrations of PGs and LTs in nasal polyps and nasal mucosa.

Samples of nasal polyps and nasal mucosa were obtained at the time of polypectomies and nasal procedures. Metabolites of arachidonic acid in tissue were determined by incubation of tissue‐homogenates with 14C‐arachidonic acid and analyses with thin‐layer chromatography and high performance liquid chromatography (HPLC). Levels of PGE2, 6‐keto‐PGF1α, throm‐boxane (Tx)B2, 15‐hydroxyeicosatetraenoic acid (HETE), LTC4, LTB4 were measured by radioimmunoassay. The predominant arachidonic acid metabolite in both nasal polyps and mucosa was 15‐HETE. The HPLC analysis showed that the predominant metabolite in nasal polyp was 15‐HETE, especially in polyps from aspirin sensitive patients. Levels of 15‐HETE and PGE2 were higher in polyps from patients with a history of allergy than from nonallergic patients. Levels of LTC4 and LTB4 in nasal polyps were determined.

The findings of this study will help to explain biochemical basis of the pathogenesis of aspirin‐sensitive nasal polyps and to develop better medical treatment for them.
Hormonal receptors in juvenile nasopharyngeal angiofibroma
Authors: Mokhtar Farag M. , Ghanimah S. E. , Ragaie A. , Saleem T. H.
Published: September 2006

Specific thermostable androgen receptors were detected in the tissues of nasopharyngeal angiofibroma. The receptors seemed to be specific with high affinity toward DHT more than testosterone. No abnormalities in serum levels of DHT, testosterone, and estradiol‐17 B could be detected in the patients studied. A concept of pathogenesis of the tumor in relation to that reported in literature recently is interpreted in the text.
Intraparotid facial nerve neurofibroma
Authors: Sullivan Michael J. , Babyak John W. , Kartush Jack M.
Published: September 2006

Neurogenic neoplasms of the intraparotid facial nerve are uncommon and are usually diagnosed intraoperatively by tissue biopsy. Fifty‐six cases of primary neurogenic neoplasms involving the intraparotid facial nerve have been reported. The majority of these have been schwannomas. A case of a solitary neurofibroma involving the main trunk of the facial nerve is presented. Schwannomas and neuro fibromas have distinct histological features which must be considered prior to the management of these tumors. The management of neurogenic tumors associated with normal facial function is a particularly difficult problem. A new approach for the diagnosis and management of neurogenic neoplasms is described utilizing electroneurography.
Etiologic factors in head and neck wound infections
Authors: Brown Bert M. , Johnson Jonas T. , Wagner Robin L.
Published: September 2006

The use of antibiotic prophylaxis does not prevent all postoperative wound infections. The records of 245 patients undergoing major head and neck surgery were reviewed to determine the cause of would infection which developed in 17 patients.

Patients with Stage IV tumors had an increased infection rate as compared to lesser stages (p<.01). Reconstruction with a myocutaneous flap was associated with an increased infection rate when compared to primary closure or split‐thickness skin graft reconstruction (p<.001). Probable errors in surgical technique were identified in 10 of 17 infected patients.

Neither weight loss, diabetes mellitus, prior radiation therapy, nor prior tracheotomy were found to increase the risk of wound infection (p>.05).

This study lends insight into factors that may potentiate the development of wound infection despite antibiotic prophylaxis. A strategy to minimize postoperative infection is offered.
Proliferative myositis of the masseter muscle
Authors: Scher Natan , Dobleman Thomas J. , Poe Dennis S. , Panje William R.
Published: September 2006

The authors present a case of a 33‐year‐old white man with a 4‐month history of a rapidly enlarging, tender, painful, circumscribed facial mass located in the masseter muscle. CT scan and fine needle aspiration cytology, coupled with clinical assessment, suggested the diagnosis of proliferative myositis. Curative intraoral excisional biopsy was done, thereby avoiding a parotidectomy incision and approach to the lesion. The pathology showed proliferative myositis. A review of the literature revealed 36 cases which indicated that proliferative myositis is a relatively rare etiology of a head and neck mass. Careful clinical, radiologic, and pathologic evaluations were required to make this unusual diagnosis and avoid any unnecessary facial mutilation in its treatment.
Fungal colonization of tracheoesophageal voice prosthesis
Authors: Izdebski Krzysztof , Ross Joel C. , Lee Scott
Published: September 2006

Laryngectomized patients fitted with tracheoesophageal (TE) puncture voice prosthesis may experience frequent failures in adequate production of neophonation. In some cases, these phonatory problems result from obstruction of the voice prosthesis with various deposits. The nature of these deposits was studied with mycologic techniques and by light and scanning electron microscopy. Candida albicans and other fungal species were identified.
Carcinoma of the nasal vestibule treated with radiation therapy
Authors: Mendenhall Nancy P. , Parsons James T. , Cassisi Nicholas J. , Million Rodney R.
Published: September 2006

Twenty‐two patients with squamous carcinoma of the nasal vestibule were treated at the University of Florida Division of Radiation Therapy with curative intent. Fifteen lesions were de novo and seven recurrent after surgery. By AJCC classification, 7 lesions were Tx or T1, 2 were T2, 2 were T3, and 11 were T4. Management of the primary tumor and regional lymphatic drainage was highly individualized. Local control was achieved in 19 out of 22 lesions. The ultimate regional lymph node control rate was 22 out of 22, although two patients required radical neck dissection after development of lymph node disease in untreated regional lymphatics. Two patients have died of cancer and three of intercurrent disease. Cosmetic results are generally excellent but may be compromised by previous surgery in recurrent lesions or tumor destruction of normal tissues in advanced lesions. Complications of treatment are minimal.
A monoclonal antibody to squamous cell carcinoma
Authors: Ranken Raymond , Kaplan Michael J. , Silverman Sol , Gottfried Toby G. , White Carmen , Fee Willard E. , Yonkovich Shirley , Boles Roger , Liu Victor
Published: September 2006

Monoclonal antibody 17.13.CI.10 is a murine IgM kappa monoclonal antibody (Mab) that stains frozen section squamous cell carcinoma (SCC) homogeneously and intensely with a sensitivity greater than 98%, including 106/107 SCC specimens from the head and neck. It was produced using a human laryngeal SCC as immunogen and screened using frozen section human tissue. Monoclonal antibody 17.13.CI.10 faintly stains the basal layer of normal squamous eptihelium, does not stain normal organ tissue other than myoepithelial cells, and reacts with few non‐SCC tumors. It, therefore, may be a useful adjunct to standard histopathologic criteria for the diagnosis of SCC. It may prove helpful in the investigation of tumor‐associated antigens. Despite major technical and immunologic problems, monoclonal antibodies to functional tissue‐specific tumor‐associated antigens have the potential to play a major role in imaging and in treatment in the future.
Autoinflation of eustachian tube in young children
Authors: Chan Kenny H. , Cantekin Erdem I. , Karnavas William J. , Bluestone Charles D.
Published: September 2006

Inflation of the middle ear has long been thought to be beneficial in the treatment of otitis media. We describe a new system of autoinflation based on a modified Valsalva technique consisting of an anesthesia mask and a flowmeter which can be used successfully by children. An objective method to record eustachian tube opening during autoinflation using frequency spectrum analysis of the ear canal sounds is also presented. Tubal opening results of this method were compared to the traditional method using preinflation and postinflation tympanometry. Study subjects included 20 adults with normal otologic findings and 35 children with middle‐ear effusion; all together 107 ears of children and adults with and without effusion were tested. Using the frequency spectrum analysis, tubal opening was recorded in 75% of adult ears and 56% of children's ears. Also, in both groups, a sound pressure peak with the same characteristic frequency around 2 kHz was associated with tubal openings. The methods described are incorporated into a clinical trial being conducted at the Children's Hospital of Pittsburgh.
The use of intradermal tests and relevance of negative controls in patients with negative or equivocal modified rast test scores to inhalant allergens
Authors: Radford Elaine R. , Berkowitz Ellis C.
Published: September 2006

Patients with suspected inhalant allergy that demonstrate a Modified RAST Test (MRT) Class 0 and 1 (equivocal) may demonstrate positive infradermal (ID) skin whealing to suspect allergens. This whealing may be due, however, to an irritant reaction from glycerin used as a preservative in the extract.

The purpose of this study is to establish the incidence of allergy as demonstrated by ID testing in patients with MRT Class 0 or 1 and to reinforce the importance of negative controls during ID testing.

Fifty‐five consecutive patients evaluated for allergy with MRT Classes 0 or 1 were studied. Intradermal testing with 1:500 wt/vol concentrations of allergenic extracts and a 2% glycerin control were performed. Thirty‐eight patients (69%) were found to have ID whealing to allergenic extracts greater than the negative glycerin controls.

We conclude that ID allergen testing of patients with MRT Class 0 or 1, when using a glycerin control, identifies additional allergens to be considered for immunotherapy.
Laryngeal papillomatosis: Clinical, histopathologic and molecular studies
Authors: Abramson Allan L. , Steinberg Bettie M. , Winkler Barbara
Published: September 2006

The clinical course and pathology of 57 patients with laryngeal papillomatosis were reviewed. Tissues from 26 patients were analyzed for human papillomavirus (HPV) DNA by Southern blot hybridization. Histopathologic evaluation of the papillomas showed no correlation with age of onset or clinical pattern of remission and recurrence. The pathology was characterized by abnormal squamous maturation with parakeratosis, retardation of superficial cell maturation, papillomatosis, and basal hyperplasia. HPV DNA was present in all lesions, with 92% containing either HPV‐6 or 11. Latent HPV DNA was detected in clinically uninvolved tissues of 11 of 14 (78.5%) patients studied. There was no correlation between HPV type, histopathology and/or clinical pattern. Despite homogeneity of pathology, the clinical expression of laryngeal HPV infection varied widely. A mechanism for the pathogenesis of laryngeal papillomatosis, based on the concept of maturational arrest, is proposed.
Extrameningeal meningiomas of the infratemporal fossa: Diagnosis and treatment
Authors: Inglis Andrew F. , Yarington C. Thomas , Bolen John
Published: September 2006

A presentation of two recently treated extrameningeal meningiomas of the infratemporal fossa will be augmented by a review of the literature on this subject. These tumors are quite rare, insidious in their presentation, and, as a benign tumor, require consideration of surgical treatment which causes the least possible functional disability. The natural history of these lesions and the surgical approach which has proved most successful in our hands will be discussed in some detail.
“OCCULT” posterior laryngeal cleft
Authors: Tucker Gabriel F. , Maddalozzo John
Published: September 2006

A previously undescribed deficiency of the posterior cricoid at the midline, wherein the mucosa overlying this deficiency is intact, is described. Clinically this presents as a variety of subglottic stenoses wherein the narrowing is primarily in the transverse subglottic diameter; the symptoms are, therefore, those of airway obstruction not laryngeal incompetence.

It is suggested that this deformity has been previously overlooked by laryngologists and pathologists by virtue of a postmortem routine in which the larynx is routinely split in the posterior midline. Demonstration in these cases was by serial horizontal section of the intact, unsplit, whole larynx.
Epithelial carcinoma of the nasal fossa
Authors: Shidnia Homayoon , Hartsough Ann B. , Weisberger Edward , Hornback Ned B.
Published: September 2006

Epithelial carcinoma of the nasal cavity and vestibule occurs relatively rarely and is primarily treated with surgical excision, radiation therapy, or a combination of these two treatment modalities. This review describes the treatment methods and results of patients seen at the Indiana University School of Medicine from 1963 through 1983 in the Departments of Radiation Oncology and Otolaryngology.

Using the medical records of 34 patients, this retrospective study reviews the clinical significance of histology, anatomical location of primary tumors, and therapeutic results, including the pattern of treatment failures of primary tumors. The results of radiation therapy alone compare favorably with the results of surgery alone. A review of the literature and this study indicate that patients receiving a combination of surgery and irradiation face a better prognosis than those who experience surgery or irradiation alone.
Eustachian tube patency detected by a photoelectric method
Authors: Yagi Nobuya , Haji Tomoyuki , Honjo Iwao
Published: September 2006

A method was developed to observe tubal orifice movement by fiberscope and detect patency of the eustachian tube by a photoelectric device (phototubometry). A highly sensitive photodiode in the external ear canal detected tube opening as increased luminosity in the tympanic cavity from a light source placed at the pharyngeal portion of the eustachian tube. The phototubometric record was superimposed on a TV screen to compare the opening in the pharyngeal portion of the eustachian tube to the tube opening during swallowing. The phototubometric records of 21 normal subjects examined by phototubometry showed the close relationship with the opened tubal lumen at the tensor veli palatini muscle (TVP) attached portion. This method is useful for examining tubalpatency and observing changes in the pharyngeal portion of the eustachian tube.
Morbidity in pediatric tonsillectomy
Authors: Linden Barry E. , Gross Charles W. , Long Thomas E. , Lazar Rande H.
Published: September 2006

Tonsillectomy is one of the most common operations performed in the pediatric age group. Previous literature on morbidity in tonsillectomy has dealt predominantly with postoperative hemorrhage. Children undergoing tonsillectomy were divided in a prospective and random fashion into eight study groups to evaluate postoperative morbidity as it relates to the surgical technique used (electrocautery, dissection, KTP laser), methods of hemostasis (electrocautery, suture ligature), and the use of postoperative antibiotics. A total of 80 children were evaluated. Using the parameters of morbidity as defined in this study, blunt dissection tonsillectomy using suture ligatures for hemostasis, without postoperative antibiotics was found to result in the least morbidity in the pediatric age group.
Demonstration of prosthetic activation of central auditory pathways using [14C]‐2‐Deoxyglucose
Authors: Evans David A. , Niparko John K. , Altschuler Richard A. , Frey Kirk A. , Miller Josef M.
Published: September 2006

The cochlear prosthesis is not applicable to patients who lack an implantable cochlea or an intact vestibulocochlear nerve. Direct electrical stimulation of the cochlear nucleus (CN) of the brain stem might provide a method for auditory rehabilitation of these patients. A penetrating CN electrode has been developed and tissue tolerance to this device demonstrated. This study was undertaken to evaluate metabolic activation of central nervous system (CNS) auditory tracts produced by such implants. Regional cerebral glucose use resulting from CN stimulation was estimated in a series of chronically implanted guinea pigs with the use of [14C]−2‐deoxyglucose (2‐DG). Enhanced 2‐DG uptake was observed in structures of the auditory tract. The activation of central auditory structures achieved with CN stimulation was similar to that produced by acoustic stimulation and by electrical stimulation of the modiolar portion of the auditory nerve in control groups. An interesting banding pattern was observed in the inferior colliculus following CN stimulation, as previously described with acoustic stimulation. This study demonstrates that functional metabolic activation of central auditory pathways can be achieved with a penetrating CNS auditory prosthesis.
Central nervous system complications associated with acute otitis media in children
Authors: Friedman Ellen M. , Mcgill Trevor J. I. , Healy Gerald B.
Published: September 2006

The purpose of this article is to review a subset of pedi‐atric patients with otitis media occurring simultaneously with meningitis and other central nervous system diseases. Of the 92 cases reviewed, 61.3% involved patients under 12 months of age. Meningitis associated with otitis media involved 91% of the patients; the remainder had nonmeningi‐tic central involvement. The most striking finding was the high incidence of Haemophilus influenza type‐B, a relatively uncommon pathogen of the middle ear. This finding highlights the need to select antibiotic coverage to adequately treat Haemophilus influenza type‐B.
Endoscopic management of frontal sinus disease
Authors: Schaefer Steven D. , Close Lanny G.
Published: September 2006

Depending on the pathologic process, the treatment of frontal sinus disease has consisted of obliteration or ablation of the sinus, or restoration of drainage into the nose. Intranasal endoscopic enlargement of the frontal recess and ostium, and removal of disease from the medial aspect of the frontal sinus offers a minimally invasive alternative to previous operations in selected patients. To better understand the indications, limitations, and potential problems with this operation, our experience with endoscopic frontal sinusotomy in 36 patients over a 30‐month period is reported. During the follow‐up period, 21 patients had complete resolution of all symptoms, 11 patients were improved but had at least one episode of sinusitis or headache post‐operatively, and 3 patients were worse, 2 of whom required frontal sinus obliteration for control of disease. Although endoscopic frontal sinusotomy appears to be a useful alternative to traditional frontal sinus procedures in selected patients, the reader is cautioned that such surgery is technically difficult and has not yet stood the test of time required of any frontal sinus operation.
Patient-related predictors of implant failure after primary total hip replacement in the initial, short- and long-terms
Authors: Johnsen S. P. , Sørensen H. T. , Lucht U. , Søballe K. , Overgaard S. , Pedersen A. B.
Published: September 2006

We examined the association between patient-related factors and the risk of initial, short- and long-term implant failure after primary total hip replacement. We used data from the Danish Hip Arthroplasty Registry between 1 January 1995 and 31 December 2002, which gave us a total of 36 984 patients. Separate analyses were carried out for three follow-up periods: 0 to 30 days, 31 days to six months (short term), and six months to 8.6 years after primary total hip replacement (long term). The outcome measure was defined as time to failure, which included re-operation with open surgery for any reason.

Male gender and a high Charlson co-morbidity index score were strongly predictive for failure, irrespective of the period of follow-up. Age and diagnosis at primary total hip replacement were identified as time-dependent predictive factors of failure. During the first 30 days after primary total hip replacement, an age of 80 years or more and hip replacement undertaken as a sequela of trauma, for avascular necrosis or paediatric conditions, were associated with an increased risk of failure. However, during six months to 8.6 years after surgery, being less than 60 years old was associated with an increased risk of failure, whereas none of the diagnoses for primary total hip replacement appeared to be independent predictors.
Total knee replacement in morbidly obese patients
Authors: Amin A. K. , Clayton R. A. E. , Patton J. T. , Gaston M. , Cook R. E. , Brenkel I. J.
Published: September 2006

The results of 41 consecutive total knee replacements performed on morbidly obese patients with a body mass index > 40 kg/m2, were compared with a matched group of 41 similar procedures carried out in non-obese patients (body mass index < 30 kg/m2). The groups were matched for age, gender, diagnosis, type of prosthesis, laterality and pre-operative Knee Society Score. We prospectively followed up the patients for a mean of 38.5 months (6 to 66). No patients were lost to follow-up. At less than four years after operation, the results were worse in the morbidly obese group compared with the non-obese, as demonstrated by inferior Knee Society Scores (mean knee score 85.7 and 90.5 respectively, p = 0.08; mean function score 75.6 and 83.4, p = 0.01), a higher incidence of radiolucent lines on post-operative radiographs (29% and 7%, respectively, p = 0.02), a higher rate of complications (32% and 0%, respectively, p = 0.001) and inferior survivorship using revision and pain as end-points (72.3% and 97.6%, respectively, p = 0.02).

Patients with a body mass index > 40 kg/m2 should be advised to lose weight prior to total knee replacement and to maintain weight reduction. They should also be counselled regarding the inferior results which may occur if they do not lose weight before surgery.
Trochleaplasty for recurrent patellar dislocation in association with trochlear dysplasia
Authors: von Knoch F. , Böhm T. , Bürgi M. L. , von Knoch M. , Bereiter H.
Published: September 2006

We investigated the clinical and radiological outcome of trochleaplasty for recurrent patellar dislocation in association with trochlear dysplasia in 38 consecutive patients (45 knees) with a mean follow-up of 8.3 years (4 to 14).

None had recurrence of dislocation after trochleaplasty. Post-operatively, patellofemoral pain, present pre-operatively in only 35 knees, became worse in 15 (33.4%), remained unchanged in four (8.8%) and improved in 22 (49%). Four knees which had no pain pre-operatively (8.8%) continued to have no pain.

A total of 33 knees were available for radiological assessment. Post-operatively, all but two knees (93.9%) had correction of trochlear dysplasia radiologically but degenerative changes of the patellofemoral joint developed in 30% (10) of the knees.

We conclude that recurrent patellar dislocation associated with trochlear dysplasia can be treated successfully by trochleaplasty, but the impact on patellofemoral pain and the development of patellofemoral osteoarthritis is less predictable. Overall, subjective patient satisfaction with restored patellofemoral stability after trochleaplasty appeared to outweigh its possible sequelae.
Vertebral Endplate Fractures: An Indicator of the Abnormal Forces Generated in the Spine After Vertebroplasty
Authors: Trout Andrew T , Kallmes David F , Layton Kennith F , Thielen Kent R , Hentz Joseph G
Published: September 2006

Vertebroplasty alters spinal biomechanics and may lead to incident vertebral fractures. The endplate localization of prevalent and incident fractures was evaluated in 86 patients. In the absence of vertebroplasty, superior endplate fractures predominate. After the procedure, inferior endplate fractures are disproportionately common in adjacent vertebrae immediately above the treated level, potentially supporting a causative relationship between vertebroplasty and incident fractures.

Introduction: To determine retrospectively whether new-onset fractures after vertebroplasty tend to cluster in the endplate immediately adjacent to the cemented vertebra.

Materials and Methods: Institutional Review Board approval and patient consent for use the use of medical records were obtained for this study. We performed a retrospective review of patients with new (incident) vertebral fractures after vertebroplasty. The median age for these patients was 72.5 years, and 58 (67.4%) were women. Fractures were diagnosed on the basis of MRI or bone scan and were catalogued based on their location within the vertebral body (superior endplate, inferior endplate, or holo-vertebral). χ2 and generalized estimating equation (GEE) analyses were used to compare the distribution of fracture subtypes among pre-existing (prevalent) and incident fractures.

Results: The patients had 313 prevalent osteoporotic vertebral fractures and were treated at 137 vertebral levels. Among prevalent fractures, superior endplate fractures predominated (57% superior, 11% inferior; p < 0.0001). After vertebroplasty, 186 incident fractures developed in these 86 patients. Seventy-seven (41%) of these incident fractures occurred adjacent to treated vertebrae. Nonadjacent, incident fractures, like prevalent fractures, occurred predominantly along superior endplate. Incident fractures immediately above treated levels, however, localized disproportionately to the inferior endplate (30% superior, 57% inferior; p < 0.0001).

Conclusions: There are an increased number of inferior endplate fractures of the vertebral body immediately cephalad to the treated level.
Independent and Combined Contributions of Cancellous and Cortical Bone Deficits to Vertebral Fracture Risk in Postmenopausal Women
Authors: Qiu Shijing , Rao D Sudhaker , Palnitkar Saroj , Parfitt A Michael
Published: September 2006

Using iliac bone histomorphometry on 78 patients with vertebral fracture and 66 healthy postmenopausal women, cortical thickness discriminated at least as well as any cancellous bone structural index between the two groups. Subjects with a deficit in both cortical and cancellous bone had much greater likelihood of fracture.

Introduction: Vertebral fracture is often attributed to disproportional loss of cancellous bone, but fracture patients may have deficits in cortical and cancellous bone. Accordingly, we examined the contribution of cortical and cancellous bone deficits, separately and together, to the likelihood of vertebral fracture.

Materials and Methods: Iliac bone histomorphometry was performed in 78 white woman with clinically apparent vertebral fracture, 66 healthy postmenopausal women, and 38 healthy premenopausal women. We measured cancellous bone volume (Cn.BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), cortical bone volume (Ct.BV/TV), and cortical thickness (Ct.Th). For each variable, a value of >1 SD below the mean in premenopausal women was treated as a putative risk factor, and its association with the presence or absence of fracture was determined by OR calculated by logistic regression and by receiver operating characteristic (ROC) curve analysis. Subsets of fracture and control subjects were separately matched for Cn.BV/TV and Ct.Th.

Results: All structural indices differed between fracture patients and controls except Ct.BV/TV. There was a weak but highly significant correlation between Cn.BV/TV and Ct.Th in the entire group (r = 0.389, r2 = 0.151 p < 0.001). Many control subjects had a high value for one of these variables and a low value for the other. Ct.Th., Cn.BV/TV, and Tb.N were all significantly associated with vertebral fracture (ORs, 4.4–5.8; ROC area under the curve [AUC], 0.74–0.85). In subjects matched for Cn.BV/TV, Ct.Th was reduced by 29% (OR, 5.0), and in subjects matched for Ct.Th, Cn.BV/TV was reduced by 27% (OR, 5.0). In patients with deficits in both cortical and cancellous bone, the ORs (28–35) were much higher.

Conclusions: Deficits in cortical bone (reduced value for Ct.Th) and in cancellous bone (reduced values for Cn.BV/TV or Tb.N) were equally effective in discriminating between subjects with and without vertebral fracture. With a deficit in both cortical and cancellous bone, the association with vertebral fracture was much stronger. Vertebral fracture is not the result of disproportionate loss of cancellous bone in the patients as a whole, although individual patients may have relatively greater deficits in either cancellous or cortical bone.
Canine Distemper Virus Induces Human Osteoclastogenesis Through NF-κB and Sequestosome 1/P62 Activation
Authors: Selby Peter L , Davies Michael , Mee Andrew P
Published: September 2006

Previous studies have implicated CDV in the pathogenesis of Paget's disease; however, there has been no direct evidence that CDV can infect human cells. We studied the effects of CDV on osteoclastogenesis in vitro and showed that CDV had a dose-dependent effect on osteoclastogenesis, through a possible mechanism involving activation of NF-κB and sequestosome 1/p62.

Introduction: Paget's disease is characterized by a dramatic increase in size and number of osteoclasts. The etiology of the disorder is still unclear; however, evidence points to either a viral infection or a genetic susceptibility or a combination of both. Previously, we have shown that canine distemper virus (CDV) RNA is present in Pagetic bone. However, the effects of CDV on human osteoclast formation in vitro have not been studied previously.

Materials and Methods: Replicate cultures (n = 5) of purified human osteoclast precursors were infected with increasing doses of CDV and cultured on dentine slices for 14 days. Osteoclasts were stained for TRACP, and the dentine slices were examined for evidence of resorption. Control cells were incubated in the absence of virus. In each case, 10 high-power microscopy fields were analyzed. Immunocytochemical analyses were performed for p65, Gab2, sequestosome 1/p62, and ubiquitin.

Results: CDV dose-dependently increased osteoclast number and size (p < 0.0001, ANOVA), and there was a concomitant increase in resorption (p < 0.0001, ANOVA). CDV infection induced nuclear translocation of p65 and led to a dramatic increase in sequestosome 1/p62 and ubiquitin expression.

Conclusions: These results provide the first conclusive proof that CDV can infect and replicate in human osteoclast precursors, raising possible zoonotic implications for CDV. The increased osteoclastogenesis is accompanied by NF-κB and sequestosome 1/p62 activation. This study provides further evidence for the possible role of paramyxoviruses in the pathogenesis of Paget's disease.
Robust and Comprehensive Analysis of 20 Osteoporosis Candidate Genes by Very High-Density Single-Nucleotide Polymorphism Screen Among 405 White Nuclear Families Identified Significant Association and Gene–Gene Interaction
Authors: Xiong Dong-Hai , Shen Hui , Zhao Lan-Juan , Xiao Peng , Yang Tie-Lin , Guo Yan , Wang Wei , Guo Yan-Fang , Liu Yong-Jun , Recker Robert R , Deng Hong-Wen
Published: September 2006

Many “novel” osteoporosis candidate genes have been proposed in recent years. To advance our knowledge of their roles in osteoporosis, we screened 20 such genes using a set of high-density SNPs in a large family-based study. Our efforts led to the prioritization of those osteoporosis genes and the detection of gene–gene interactions.

Introduction: We performed large-scale family-based association analyses of 20 novel osteoporosis candidate genes using 277 single nucleotide polymorphisms (SNPs) for the quantitative trait BMD variation and the qualitative trait osteoporosis (OP) at three clinically important skeletal sites: spine, hip, and ultradistal radius (UD).

Materials and Methods: One thousand eight hundred seventy-three subjects from 405 white nuclear families were genotyped and analyzed with an average density of one SNP per 4 kb across the 20 genes. We conducted association analyses by SNP- and haplotype-based family-based association test (FBAT) and performed gene–gene interaction analyses using multianalytic approaches such as multifactor-dimensionality reduction (MDR) and conditional logistic regression.

Results and Conclusions: We detected four genes (DBP, LRP5, CYP17, and RANK) that showed highly suggestive associations (10,000-permutation derived empirical global p ≤ 0.01) with spine BMD/OP; four genes (CYP19, RANK, RANKL, and CYP17) highly suggestive for hip BMD/OP; and four genes (CYP19, BMP2, RANK, and TNFR2) highly suggestive for UD BMD/OP. The associations between BMP2 with UD BMD and those between RANK with OP at the spine, hip, and UD also met the experiment-wide stringent criterion (empirical global p ≤ 0.0007). Sex-stratified analyses further showed that some of the significant associations in the total sample were driven by either male or female subjects. In addition, we identified and validated a two-locus gene–gene interaction model involving GCR and ESR2, for which prior biological evidence exists. Our results suggested the prioritization of osteoporosis candidate genes from among the many proposed in recent years and revealed the significant gene–gene interaction effects influencing osteoporosis risk.
Bone Density Ligand, Sclerostin, Directly Interacts With LRP5 but Not LRP5G171V to Modulate Wnt Activity
Authors: Ellies Debra L , Viviano Beth , McCarthy John , Rey Jean-Philippe , Itasaki Nobue , Saunders Scott , Krumlauf Robb
Published: September 2006

We compared and contrasted the mechanism of action for the cysteine knot protein subfamily, Wise and Sost (Sclerostin). Our data suggest that functional interactions between Sost or Wise and LRP5/LRP6 have the potential to regulate bone deposition by modulating the Wnt pathway.

Introduction: The human disease sclerosteosis exhibits an increase in bone mass thought to be caused by hyperactive osteoblasts. Sclerostin, SOST, the gene affected in this disease, has been postulated to exert its activity by functioning as a BMP antagonist. However, recent evidence indicates that SOST is highly related to Wise, which can also modulate the Wnt pathway by binding to LRP5 and LRP6.

Materials and Methods: For this study, we used cell culture to test the BMP and Wnt activity function of both Wise and Sost. In addition, we used Xenopus in vivo Wnt assays along with Xenopus in vitro Wnt assays to support our cell culture results. Epitope tagged cell supernatants containing either Sost or soluble mutant or wildtype LRP5/LRP6 were used for immunoprecipitation. Sost immunoprecipitation results were confirmed in vivo using cell culture. Finally, to support our in vitro data, we co-localized Sost, Wise, LRP5, and LRP6 in mouse long bone sections.

Results: In this study, we report in vitro and in vivo evidence to show that Sost physically interacts with Lrp5 and Lrp6 and inhibits the canonical Wnt signaling pathway. Furthermore, using in vitro and in vivo assays, we showed that a variant of LRP5 (LRP5G171V) known to cause the human high bone mass (HBM) trait and a homologous change in LRP6 (LRP6G158V) abolished protein interactions with Sost. We used variants of Sost amino acids to further identify the contact points between Sost and LRP6. In Xenopus and mammalian cell culture assays, we showed that SOST is able to attenuate Wnt signaling and that this attenuation can be rescued by the addition of α-Sost antibodies or by the introduction of single amino acid substitution that alter its binding to LRP6. Sost differs from Wise in that it is unable to stimulate Wnt signaling. Using immunohistochemistry, we found that Sost and Wise are co-localized to osteoblasts, along with LRP5 and LRP6.

Conclusions: Our data suggest that functional interactions between Sost or Wise and LRPs have the potential to regulate bone deposition by modulating Wnt signaling.
Antibodies to Stem Cell Marker Antigens Reduce Engraftment of Hematopoietic Stem Cells
Authors: Gilner Jennifer B. , Walton William G. , Gush Kimberly , Kirby Suzanne L.
Published: September 2006

Hematopoietic stem cells (HSCs) have enormous potential for use in transplantation and gene therapy. However, the frequency of repopulating HSCs is often very low; thus, highly effective techniques for cell enrichment and maintenance are required to obtain sufficient cell numbers for therapeutic use and for studies of HSC physiology. Common methods of HSC enrichment use antibodies recognizing HSC surface marker antigens. Because antibodies are known to alter the physiology of other cell types, we investigated the effect of such enrichment strategies on the physiology and lineage commitment of HSCs. We sorted HSCs using a method that does not require antibodies: exclusion of Hoechst 33342 to isolate side population (SP) cells. To elucidate the effect of antibody binding on this HSC population, we compared untreated SP cells with SP cells treated with the Sca-1+c-Kit+Lin− (SKL) antibody cocktail prior to SP sorting. Our findings revealed that HSCs incubated with the antibody cocktail had decreased expression of the stem cell-associated genes c-Kit, Cd34, Tal-1, and Slamf1 relative to untreated SP cells or to cells treated with polyclonal isotype control antibodies. Moreover, SKL antibodies induced cycling in SP cells and diminished their ability to confer long-term hematopoietic engraftment in lethally irradiated mice. Taken together, these data suggest that antibody-based stem cell isolation procedures can have negative effects on HSC physiology.
N-Glycolylneuraminic Acid Xenoantigen Contamination of Human Embryonic and Mesenchymal Stem Cells Is Substantially Reversible
Authors: Heiskanen Annamari , Satomaa Tero , Tiitinen Sari , Laitinen Anita , Mannelin Sirkka , Impola Ulla , Mikkola Milla , Olsson Cia , Miller-Podraza Halina , Blomqvist Maria , Olonen Anne , Salo Hanna , Lehenkari Petri , Tuuri Timo , Otonkoski Timo , Natunen Jari , Saarinen Juhani , Laine Jarmo
Published: September 2006

Human embryonic and mesenchymal stem cell therapies may offer significant benefit to a large number of patients. Recently, however, human embryonic stem cell lines cultured on mouse feeder cells were reported to be contaminated by the xeno-carbohydrate N-glycolylneuraminic acid (Neu5Gc) and considered potentially unfit for human therapy. To determine the extent of the problem of Neu5Gc contamination for the development of stem cell therapies, we investigated whether it also occurs in cells cultured on human feeder cells and in mesenchymal stem cells, what are the sources of contamination, and whether the contamination is reversible. We found that N-glycolylneuraminic acid was present in embryonic stem cells cultured on human feeder cells, correlating with the presence of Neu5Gc in components of the commercial serum replacement culture medium. Similar contamination occurred in mesenchymal stem cells cultured in the presence of fetal bovine serum. The results suggest that the Neu5Gc is present in both glycoprotein and lipid-linked glycans, as detected by mass spectrometric analysis and monoclonal antibody staining, respectively. Significantly, the contamination was largely reversible in the progeny of both cell types, suggesting that decontaminated cells may be derived from existing stem cell lines. Although major complications have not been reported in the clinical trials with mesenchymal stem cells exposed to fetal bovine serum, the immunogenic contamination may potentially be reflected in the viability and efficacy of the transplanted cells and thus bias the published results. Definition of safe culture conditions for stem cells is essential for future development of cellular therapies.
Analysis of Immunotoxicity by Enumeration of Antibody‐Producing B Cells
Authors: Anderson Stacey E. , Munson Albert E. , Meade Barbara J.
Published: September 2006

The plaque‐forming cell (PFC) assay measures the humoral immune response mediated by the concerted actions of antigen‐presenting cells, T lymphocytes, and B lymphocytes. The most common form of the plaque method is used for the detection of murine primary IgM antibodies directed against the T cell–dependent sheep red blood cell (sRBC) surface antigens. Research has shown that the PFC response to sRBC is not only an excellent monitor of the primary effector function of the B cell, but that it may be the most sensitive immune parameter currently available to identify chemical perturbation. Several modifications and variations of the PFC assay are described in detail in this unit.
The Reporter Antigen Popliteal Lymph Node Assay
Authors: Nierkens Stefan , Pieters Raymond
Published: September 2006

Many chemicals, including drugs and environmental pollutants, may have the intrinsic capacity to stimulate or dysregulate immune responses. These responses may create considerable problems for exposed subjects in terms of development of autoimmunity or hypersensitivity reactions. The popliteal lymph node assay (PLNA) provides a suitable tool to assess the immunostimulating potential of chemicals and might be a potential candidate as a screening tool in immunotoxicological hazard identification. The use of so‐called reporter antigens (RA) in this assay additionally enables differentiation between immunosensitizing (sensitizers), immunostimulating (irritants), and innocent chemicals. In the RA‐PLNA, the compound of interest is injected into the hind footpad together with a nonsensitizing dose of the RA. After 6 to 8 days, RA‐specific responses are monitored in the draining PLN by measuring RA‐specific antibody formation, cytokine secretion, and shifts in immune cell numbers. Hence, this simple and straightforward assay provides immunologically relevant information about the immunomodulating properties of a chemical.
Endoglycosidase and Glycoamidase Release of N‐Linked Oligosaccharides
Authors: Freeze Hudson H. , Kranz Christian
Published: September 2006

Nearly all proteins entering the lumen of the endoplasmic reticulum (ER) become glycosylated en route to a cellular organelle, the plasma membrane, or the extracellular space. Many glycans can be attached to proteins, but the most common are the N‐linked oligosaccharides. These chains are added very soon after a protein enters the ER, but they undergo extensive remodeling (processing), especially in the Golgi. Processing changes the sensitivity of the N‐glycan to enzymes that cleave entire sugar chains or individual monosaccharides, which also changes the migration of the protein on SDS gels. These changes can be used to indicate when a protein has passed a particular subcellular location. This unit details some of the methods used to track a protein as it traffics from the ER to the Golgi toward its final location.
Dog EEG for Wake‐Promotion Studies
Authors: Parmentier Régis , Bricout Denis , Brousseau Emmanuel , Giboulot Thierry
Published: September 2006

Described in this unit is a protocol for investigating the wake‐promoting activity of new chemical entities (NCEs) in dog. The experimental approach is based on scoring of sleep/wake stages in animals implanted with a telemetry device for recording EMG and cortical EEG signals. A major advantage of this procedure is that it is conducted in nontethered animals, limiting possible bias and complications encountered with conventional recording systems. In this procedure, polygraphic recording is conducted using four implanted beagles. Results of studies with modafinil, a wake‐promoting agent, are described to demonstrate the utility of this test procedure.
In Vivo Animal Models: Quantitative Models Used for Identifying Antibacterial Agents
Authors: Fernandez Jeffrey
Published: September 2006

Determining the in vivo bactericidal potential of novel agents is critical for selecting drug candidates. Described in this unit are two protocols that measure the amount of bacterial killing under very different conditions. The mouse pouch protocol is a rapid assay that provides efficacy data in an acute infection, whereas the rabbit tissue cage protocol assesses the ability of a compound to eradicate bacteria in a chronic abscess infection. Data from these tests, along with those from other qualitative and quantitative assays, are important for defining antibacterial efficacy in vivo.
In Vitro Antibacterial Resistance Selection and Quantitation
Authors: Young Katherine
Published: September 2006

The study of resistance is vital to the discovery and development of new antibacterial agents. Consider the case where the resistance frequency (the proportion of cells within a population that exhibit a resistance phenotype to the agent under study) of an agent is such that, in an average infection, a mutant resistant to the agent is likely to already exist. This compound will not be useful in the clinic as a single agent, since this mutant can survive its administration, leading to potential clinical failure. This unit provides protocols for determining the frequency and rate of resistance to antibacterial agents, as well as generating resistant mutants for mechanism of action determinations or for use in antibacterial discovery or development programs.
Fluorescence Quenching Methods to Study Lipid‐Protein Interactions
Authors: Carney Joanne , East J. Malcolm , Mall Sanjay , Marius Phedra , Powl Andrew M. , Wright J. Neville , Lee Anthony G.
Published: September 2006

This unit describes how fluorescence quenching methods can be used to determine binding constants for phospholipids binding to intrinsic membrane proteins. Reconstitution of a Trp‐containing intrinsic membrane protein with bromine‐containing phospholipids leads to quenching of the Trp fluorescence of the protein; the extent of quenching depends on the strength of binding of the phospholipid to the protein. Protocols are included for the synthesis of bromine‐containing phospholipids from phospholipids containing carbon‐carbon double bonds in their fatty acyl chains and for the reconstitution of membrane proteins into bilayers containing bromine‐containing phospholipids. Details are included on data analysis, including equations and software that can be used for fitting the fluorescence quenching data.