Mitochondrial dysfunction in the skeletal muscle of a mouse model of Rett syndrome (RTT): implications for the disease phenotype.

Rett syndrome (RTT) is a severe neurodevelopmental disorder, predominantly caused by mutations in the X-linked Methyl-CpG-binding protein 2 (MECP2) gene. Patients present with numerous functional deficits including intellectual disability and abnormalities of movement. Clinical and biochemical features may overlap with those seen in patients with primary mitochondrial respiratory chain disorders. In the late stages of the disorder, patients suffer from motor deterioration and usually require assisted mobility. Using a mouse model of RTT (Mecp2(tm1Tam)), we studied the mitochondrial function in the hind-limb skeletal muscle of these mice. We identified a reduction in cytochrome c oxidase subunit I (MTCO1) at both the transcript and protein level, in accordance with our previous findings in RTT patient brain studies. Mitochondrial respiratory chain (MRC) enzyme activity of complexes II+III (COII+III) and complex IV (COIV), and glutathione (GSH) levels were significantly reduced in symptomatic mice, but not in the pre-symptomatic mice. Our findings suggest that mitochondrial abnormalities in the skeletal muscle may contribute to the progressive deterioration in mobility in RTT through the accumulation of free radicals, as evidenced by the decrease in reduced glutathione (GSH). We hypothesise that a diminution in GSH leads to an accumulation of free radicals and an increase in oxidative stress. This may impact on respiratory chain function and contribute in part to the progressive neurological and motor deterioration seen in the Mecp2-mutant mouse. Treatment strategies aimed at restoring cellular GSH levels may prove to be a novel target area to consider in future approaches to RTT therapies.

Intra-operative frozen section analysis for suspected early-stage ovarian cancer: 11 years of Gateshead Cancer Centre experience.

OBJECTIVE: In centres in which intra-operative frozen section (FS) analysis is not performed, 'apparent' early-stage ovarian cancer diagnosed after surgery on paraffin section may require further restaging laparotomy or adjuvant chemotherapy. Previous studies on FS analysis have reported high sensitivity, specificity and overall accuracy. The objective of this article is to present the largest published dataset on the accuracy of FS analysis over an 11-year period from a single institution. DESIGN: Diagnostic test accuracy. SETTING: Northern Gynaecological Oncology Centre and Department of Cellular Pathology, Gateshead, UK. POPULATION: 1439 intra-operative FS analyses performed between January 2000 and December 2010 for suspected ovarian cancer. METHODS: Prospectively collected data on FS analysis were compared with gold standard paraffin section. MAIN OUTCOME MEASURES: Sensitivity, specificity, likelihood ratios and post-test probability. RESULTS: The overall sensitivity and specificity of FS analysis were 91.2% and 98.6%, respectively. Positive and negative likelihood ratios were 64.7% and 0.09%, respectively. The pre-test probability of an ovarian tumour being borderline or malignant was 45.8%. When FS analysis was reported to be positive, the post-test probability increased to 98% (confidence interval, 97-99%). Conversely, when FS analysis was reported to be negative, the post-test probability decreased to 7% (confidence interval, 6-9%). The majority of false test results were either borderline tumours or of mucinous differentiation. CONCLUSIONS: Intra-operative FS analysis has excellent diagnostic test accuracy and assists gynaecological oncologists to perform the appropriate surgery in 95% of cases, thereby preventing the morbidity of surgical staging in benign cases and the morbidity of restaging procedures or chemotherapy in early-stage malignant tumours.

Lost in translation: translational interference from a recurrent mutation in exon 1 of MECP2.

BACKGROUND: Rett syndrome (RTT) is an X linked neuro-developmental disorder affecting mostly girls. Mutations in the coding region of MECP2 are found in 80% of classic RTT patients. Until recently, the region encoding MECP2 was believed to comprise exons 2, 3, and 4 with the ATG start site located at the end of exon 2 (MeCP2_e2). METHODS: Recent reports of another mRNA transcript transcribed from exon 1 (MeCP2_e1) prompted us to screen exon 1 among RNA samples from 20 females with classic or atypical RTT. RESULTS: A previously reported 11 base pair deletion in exon 1 was detected in one subject with a milder phenotype. Although RNA expression for both protein isoforms was detected from the mutant allele, evaluation of MeCP2 protein in uncultured patient lymphocytes by immunocytochemistry revealed that MeCP2 protein production was restricted to only 74-76% of lymphocytes. X chromosome inactivation studies of genomic DNA revealed similar XCI ratios at the HUMARA locus (73:27 with HpaII and 74:26 with McrBC). We have demonstrated that translation but not transcription of the MeCP2_e2 isoform is ablated by the 11 nucleotide deletion, 103 nucleotides upstream of the e2 translation start site. CONCLUSIONS: These findings reveal that nucleotides within the deleted sequence in the 5'-UTR of the MeCP2_e2 transcript, while not required for transcription, are essential for translation.

Mucous cyst of the alar base: a rare complication following rhinoplasty.

Mucous cyst formation following rhinoplasty is a rare complication and typically presents late. Previously reported cases invariably involve the dorsum of the nose or nasal tip. We present a case where this unusual lesion was found to involve the alar base.

The genetic background modifies the spontaneous and X-ray-induced tumor spectrum in the Apc1638N mouse model.

The effect of the genetic background on the tumor spectrum of Apc1638N, a mouse model for attenuated familial adenomatous polyposis (FAP), has been investigated in X-irradiated and untreated F1 hybrids between C57BL/6JIco-Apc1638N (B6) and A/JCrIBR (A/J), BALB/cByJIco (C) or C3H/HeOuJIco (C3). Similar to the ApcMin model, the Apc1638N intestinal tumor multiplicity seems to be modulated by Mom1. Moreover, several additional (X-ray-responsive) modifier loci appear also to affect the Apc1638N intestinal tumor number. The genetic background did not significantly influence the number of spontaneous desmoids and cutaneous cysts in Apc1638N. In general, X-irradiation increased the desmoid multiplicity in Apc1638N females but had no effect in males. The opposite was noted for the cyst multiplicity after X-rays. Surprisingly, X-irradiated CB6F1-Apc1638N females were highly susceptible to the development of ovarian tumors, which displayed clear loss of the wild-type Apc allele.

Intestinal tumorigenesis in the Apc1638N mouse treated with aspirin and resistant starch for up to 5 months.

The Apc1638N mouse model, which carries a targeted mutant allele within the adenomatous polyposis (Apc) gene and develops intestinal tumours spontaneously, predominantly in the small bowel, was used to investigate the effects of two potential chemopreventive agents, aspirin and alpha-amylase resistant starch (RS). Heterozygous Apc+/Apc1638N mice were fed semi-purified diets rich in animal fat, animal proteins and sucrose and low in dietary fibre (Western style diets) from approximately 6 weeks up to 6 months of age. Two of the diets contained aspirin (300 mg/kg diet) and two RS (1:1 mixture of raw potato starch: Hylon VII at 200 g/kg diet) in a 2 x 2 factorial design. A fifth treatment group were fed a conventional rodent chow diet. The mice fed the Western style diets became almost three times as fat as the chow-fed mice but this did not affect tumour yield. Treatment with RS resulted in significantly more intestinal tumours whereas aspirin alone had no effect. However, there was a significant aspirin x RS interaction, which suggests that aspirin could prevent the small intestine tumour-enhancing effects of RS in this Apc-driven tumorigenesis model. The possibility that large amounts of purified forms of resistant starch may have adverse effects within the small bowel is a novel observation that requires further investigation since greater intakes of starchy foods (and of RS) are being encouraged as a public health measure in compensation for reduced dietary fat intake. However, it remains possible that any increased risk is restricted to carriers of germline mutations in APC.

New monoclonal antibodies to the T cell antigens CD4 and CD8. Production and characterization in formalin-fixed paraffin-embedded tissue.

We have generated a recombinant protein representing part of the CD4 molecule and a peptide representing an epitope of predicted high antigenicity on the CD8 molecule and employed these to generate mouse monoclonal antibodies using standard hybridoma protocols. The extracellular domain of the CD4 molecule was obtained by reverse transcription of mRNA from peripheral blood lymphocytes followed by polymerase chain reaction. The amplified gene fragment was cloned into an expression vector to allow a histidine-tagged fusion protein to be produced in Escherichia coli. Purified fusion protein was used to immunize mice. The CD8 monoclonal antibody was raised against a peptide consisting of 13 amino acids within the carboxyl-terminal region of the CD8 cytoplasmic domain. The antibodies showed appropriate reactivity on Western blotting. By heat pretreatment, these antibodies have been shown to be highly effective on paraffin-embedded tissue. In normal lymphoid tissue, the expected distribution of CD4 and CD8 lymphocytes was observed. In a series of 16 T cell lymphomas and B cell lymphomas, immunostaining results were compared with those obtained using reagents effective only in frozen tissue. A high degree of correlation was observed. These results suggest that NCL-CD4 and NCL-CD8 may be of value in the characterization of T cell disorders.

Intestinal and extra-intestinal tumor multiplicities in the Apc1638N mouse model after exposure to X-rays.

Seven-week-old Apc1638N mice were exposed to a single dose of 5 Gy total-body X-irradiation resulting in a 8-fold increase in the number of intestinal tumors and a reduction of the lifespan to an average of 6 months. The distribution of tumors along the intestinal tract as well as the adenoma/carcinoma ratio, were similar between non-irradiated and irradiated animals. Semi-quantitative PCR analysis of intestinal-tumor DNA revealed that 10 out of 14 tumors had lost the wild-type Apc allele. However, in contrast to spontaneous Apc1638N intestinal tumors in which the LOH event at the Apc locus involves the entire chromosome 18 (1), in 6 out of 10 tumors derived from X-irradiated animals the Apc loss is associated with only a partial intrachromosomal deletion. The remaining tumors have lost all chromosome 18 markers tested. In addition to the intestinal tumors, female Apc1638N mice are susceptible to the development of mammary tumors. Upon X-irradiation, Apc1638N mice show a striking 15-fold increase in mammary tumors. Moreover, Apc1638N mice spontaneously develop other extra-intestinal neoplasia, such as desmoid-like lesions similar to those associated with familial adenomatous polyposis (FAP), the human syndrome caused by germline mutations in the APC gene. Spontaneous desmoid growth is sex-dependent, as male Apc1638N mice develop 3-fold more desmoids than female mice. Interestingly, X-irradiation seemed to increase the number of desmoids per animal nearly twofold only in female Apc1638N mice. Five out of 9 desmoids found in Apc1638N mice exposed to X-ray displayed loss of the wild-type Apc allele.

The effects of different sampling techniques on smear quality and the diagnosis of cytological abnormalities in cervical screening.

A major cause of false-negative cervical smear is sampling error. We examined the results obtained with three different instruments in 126,608 smears from general practitioners. The spatula/brush combination yielded the highest proportion of smears showing cytological abnormalities, and the Cervex brush the lowest. Although not a randomized study, this paper highlights the shortcomings of the Cervex brush. We postulate a mechanical deficiency. Diagnostic accuracy rather than a high proportion of good quality smears should dictate the choice of instrument.

Radiographic findings of extremity tuberculosis in childhood: back to the future?

Since 1985, there has been an increase in the incidence of skeletal tuberculosis. Although this recent increase may prove to be temporary, familiarity with the protein manifestations of the disease is essential. The authors studied radiographs of 45 children in whom tuberculosis of the extremities had been diagnosed between 1937 and July 1991. Disease involved the hip in 18 cases; the knee in 17; the ankle in five; the shoulder in two; the midfoot in two; and the elbow, wrist, and hands in one each. Radiographic findings included joint effusion, periarticular osteopenia, joint space narrowing, cortical irregularity, lytic lesions, periosteal new bone formation, and advanced epiphyseal maturity. When the hip was involved, subluxation was a common finding. There is no single pathognomonic finding with which to make the diagnosis of skeletal tuberculosis. Clinical information may be helpful.

Effect of mixing allergenic extracts containing Helminthosporium, D. farinae, and cockroach with perennial ryegrass.

Allergenic extracts used for immunotherapy often contain mixes of different allergens. Studies have shown that certain allergenic extracts contain enzymes that can cause a decrease in grass pollen allergenicity when mixed with grass extracts. Glycerin and buffered saline with phenol (BSP) extracts containing Helminthosporium interseminatum, D. farinae, and cockroach were mixed with perennial ryegrass extracts and analyzed 7, 30, 60, and 90 days after mixing to determine the mixing effect of these extracts on the ryegrass pollen potency. Analysis was performed using RAST inhibition, SDS-PAGE and, to determine biologic potency, a quantitative intradermal skin test technique. All tests showed significant decreases in ryegrass potency when mixed with Helminthosporium and cockroach. This decrease was not seen with the D. farinae mix. Glycerin seemed to have some protective effect. Even in the situation showing the most decrease in ryegrass potency (Helminthosporium mixed with ryegrass in a BSP extract), the resulting extract still contained an estimated biologic potency of 10,000 AU/mL. This may explain why such mixes, which have been used clinically for many years, appear to give adequate therapeutic results. The study also suggest that clinicians may be well advised not to mix grass pollen extracts with extracts that may contain proteolytic enzymes.

Standardization of allergenic extracts: a manufacturer's perspective.

The current procedure for standardizing allergenic extracts utilizes quantitative skin testing techniques and in vitro techniques. A reference preparation is selected and tested using a sensitive intradermal skin test technique to determine allergy units. Subsequent batches of standardized extracts are compared to the reference preparation using in vitro techniques. The standardization program has accelerated over the past few years and all indications are that it will continue to do so in the future.

An expandable prosthesis for stabilization of the infant mediastinum following pneumonectomy.

One possible complication in infant pneumonectomy is mediastinal shift that can fatally kink or compress airways and vessels. Rigid prostheses have been used to prevent these problems; however, they cannot be adjusted as the child grows. We report a case of expandable prosthesis implantation in a 24-day-old infant. During the 18 months postimplantation, the prosthesis was periodically injected with a saline/contrast solution to maintain the mediastinum in a midline position as the child grew. At 24-month follow-up the prosthesis was still in place, and midline position of the mediastinum maintained.

High resolution ultrasound with Doppler: a diagnostic adjunct in orbital and ocular lesions in children.

Twenty-six infants and children with orbital and ocular pathology were examined with ultrasound (US) utilizing real-time imaging and Duplex Pulsed Doppler evaluation. Twenty-two of these patients underwent concurrent orbital computed tomography (CT) and two had magnetic resonance imaging (MRI). Orbital and periorbital lesions included hemangioma, dermoid, lymphangioma, rhabdomyosarcoma, encephalocoele and abscess. Ocular lesions included infection, trauma, retinal detachment, retinoblastoma, Coat's disease, and persistent hyperplastic primary vitreous. High resolution US with Doppler provided unique diagnostic information in patients with penetrating ocular trauma, orbital and periorbital masses, and intraocular structural abnormalities. High resolution US examination of the eye and periorbital tissues is readily performed using widely available equipment and often delineates subtle structural abnormalities not shown by CT or MRI.

Eosinophilia-myalgia syndrome: findings on chest radiographs in 18 patients.

Findings on chest radiographs of 18 patients with the eosinophilia-myalgia syndrome were compared and correlated with peak eosinophil counts. Nine patients had normal chest radiographs. Of the nine patients with abnormal chest radiographs, three had fine, irregular linear opacities in the lungs (the opacities were most noticeable at the bases); three had similar irregular linear opacities and pleural effusions; and three had pleural effusions and confluent opacities. One patient demonstrated an enlarged heart; no pulmonary edema was seen in this patient or in any others. Although the mean recorded eosinophil cell counts were higher in those patients with abnormal chest radiographs (6,340 vs 5,454/mm3 [6.3 vs 5.4 x 10(9)/L]), the difference was not statistically significant.

Ultrasound in advanced pediatric osteomyelitis. A report of 5 cases.

The use and reporting of ultrasound for the evaluation of pediatric osteomyelitis is minimal. However, ultrasound is noninvasive and does not employ ionizing radiation, and it is less expensive than other methods available. Consequently, its application, if contributory, would be desirable in evaluating pediatric osteomyelitis. We examined five patients ranging from 2-weeks to ten-years-old with ultrasound after bony abnormalities were found on plain film. In all cases ultrasound was valuable in assessing the type and location of abnormality (4 cases of osteomyelitis, 1 case of discitis), but unhelpful in determining the age of disease.

Review of radiologic and clinical findings in the recombinant 8 syndrome.

The findings, including radiographic findings, in recombinant 8 syndrome, a rare syndrome in patients with an unbalanced partial duplication/partial deletion of chromosome 8, are described. In addition, the carrier status and heritability are discussed.

Porcelain gallbladder in a child: a case report and review.

Calcification of the gallbladder wall (porcelain gallbladder; PGB) is a rare form of gallbladder disease not previously described in a child. A 10-year-old girl is presented with PGB that was discovered incidentally during intravenous urography. Computed tomography localized the calcification to the gallbladder wall. Cholecystectomy was performed due to the associated increased incidence of biliary tract carcinoma reported in adult patients. The etiology, diagnosis, and management of PGB and its significance in a pediatric patient are discussed.