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Progressive skeletal disease accounts for some of the most debilitating complications of type 1 Gaucher disease. In this 48-month, prospective, non-randomized, open-label study of the effect of enzyme replacement therapy on bone response, 33 imiglucerase-naïve patients (median age 43 years with one or more skeletal manifestations such as osteopenia, history of bone crisis, or other documented bone pathology) received imiglucerase 60 U/kg/2 weeks. Substantial improvements were observed in bone pain (BP), bone crises (BC), and bone mineral density (BMD). Improvements in BP were observed at 3 months (p < 0.001 vs baseline) and continued progressively throughout the study, with 39% of patients reporting pain at 48 months vs 73% at baseline. Eleven of the 13 patients with a pre-treatment history of BC had no recurrences. Biochemical markers for bone formation increased; markers for bone resorption decreased. Steady improvement of spine and femoral neck BMD, measured using dual-energy X-ray absorptiometry was noted. Mean Z score for spine increased from -0.72 +/- 1.302 at baseline to near-normal levels (-0.09 +/- 1.503) by month 48 (p = 0.042) and for femoral neck from -0.59 +/- 1.352 to -0.17 +/- 1.206 (p = 0.035) at month 36. This increase was sustained at 48 months. With imiglucerase treatment, patients should anticipate resolution of BC, rapid improvement in BP, increases in BMD, and decreased skeletal complications.

Fatty acyl-CoAs participate in numerous cellular processes. This article describes a method for the quantitation of subpicomole amounts of long-chain and very-long-chain fatty acyl-CoAs by reverse-phase LC combined with electrospray ionization tandem mass spectrometry in positive ion mode with odd-chain-length fatty acyl-CoAs as internal standards. This method is applicable to a wide range of species [at least myristoyl- (C14:0-) to cerotoyl- (C26:0-) CoA] in modest numbers of cells in culture ( approximately 10(6)-10(7)), with analyses of RAW264.7 cells and MCF7 cells given as examples. Analysis of these cells revealed large differences in fatty acyl-CoA amounts (12 +/- 1.0 pmol/10(6) RAW264.7 cells vs. 80.4 +/- 6.1 pmol/10(6) MCF7 cells) and subspecies distribution. Very-long-chain fatty acyl-CoAs with alkyl chain lengths > C20 constitute <10% of the total fatty acyl-CoAs of RAW264.7 cells versus >50% for MCF7 cells, which somewhat astonishingly contain approximately as much C24:0- and C26:0-CoAs as C16:0- and C18:0-CoAs and essentially equal amounts of C26:1- and C18:1-CoAs. This simple and robust method should facilitate the inclusion of this family of compounds in "lipidomics" and "metabolomics" studies.

Fabry disease (FD) is an X-linked lysosomal storage disease caused by alpha-galactosidase A deficiency. The Fabry Registry is a global clinical effort to collect longitudinal data on FD. In the past, most "carrier" females were usually thought to be clinically unaffected. A systematic effort has been made to enroll all FD females, regardless of symptomology. Of the 1077 enrolled females in the Registry, 69.4% had symptoms and signs of FD. The median age at symptom onset among females was 13 years, and even though 84.1% had a positive family history, the diagnosis was not made until a median age of 31 years. Twenty percent experienced major cerebrovascular, cardiac, or renal events, at a median age of 46 years. Among adult females with estimated glomerular filtration rate (eGFR) data (N=638), 62.5% had an eGFR <90 ml/min/1.73 m2 and 19.0% had eGFR <60 ml/min/1.73 m2. Proteinuria 300 mg/day was present in 39.0% of females, and 22.2% had >1 gram/day. Quality of life (QoL), as measured by the SF-36((R)) survey, was impaired at a later age than in males, but both genders experience significantly impaired QoL from the third decade of life onward. Thus, females with FD have a significant risk for major organ involvement and decreased QoL. Females should be regularly monitored for signs and symptoms of FD, and considered for enzyme replacement therapy.

The NF2 tumor suppressor gene on chromosome 22 is a member of the protein 4.1 family of cytoskeletal elements. A number of single- and multiple-tumor phenotypes have been linked to alterations of NF2 since its characterization in 1993. We present a meta-analysis of 967 constitutional and somatic NF2 alterations from 93 published reports, along with 59 additional unpublished events identified in our laboratory and 115 alterations identified in clinical samples submitted to the Massachusetts General Hospital (MGH) Neurogenetics DNA Diagnostic Laboratory. In total, these sources defined 1,070 small genetic changes detected primarily by exon scanning, 42 intragenic changes of one whole exon or larger, and 29 whole gene deletions and gross chromosomal rearrangements. Constitutional single-exon events (N=422) were significantly more likely to be nonsense or splice site changes than somatic events (N=533), which favored frameshift changes (chi(2) test; P<0.001). Somatic events also differed markedly between tumors of different pathology, most significantly in the tendency of somatic events in meningiomas to lie within the 5' FERM domain of the transcript (Fisher's exact test; P<0.01 in comparison to schwannomas) with a complete absence of mutations in exons 14 and 15. There was no statistically significant difference in mutation type or exon distribution between published constitutional events and those found by the clinical laboratory. Less than 10% of all published and unpublished small alterations are nontruncating (N=63) and these changes are clustered in exons 2 and 3, suggesting that this region may be especially crucial to tumor suppressor activity in the protein., (Published 2006 Wiley-Liss, Inc.)

Fabry disease is an X-linked metabolic storage disorder due to the deficiency of lysosomal alpha-galactosidase A, and the subsequent accumulation of glycosphingolipids, primarily globotriaosylceramide, throughout the body. Males with classical Fabry disease develop early symptoms including pain and hypohidrosis by the second decade of life reflecting disease progression in the peripheral and autonomic nervous systems. An insidious cascade of disease processes ultimately results in severe renal, cardiac, and central nervous system complications in adulthood. The late complications are the main cause of late morbidity, as well as premature mortality. Disease presentation in female heterozygotes may be as severe as in males although women may also remain asymptomatic. The recent introduction of enzyme replacement therapy to address the underlying pathophysiology of Fabry disease has focused attention on the need for comprehensive, multidisciplinary evaluation and management of the multi-organ system involvement. In anticipation of evidence-based recommendations, an international panel of physicians with expertise in Fabry disease has proposed guidelines for the recognition, evaluation, and surveillance of disease-associated morbidities, as well as therapeutic strategies, including enzyme replacement and other adjunctive therapies, to optimize patient outcomes.

Objectives: Norrie disease is an X-linked recessive disorder in which patients are born blind and develop sensory hearing loss in adolescence. The hearing loss associated with Norrie disease has been shown in a genetically altered knockout mouse to involve dysfunction of the stria vascularis; most other structures are preserved until the later stages of the disease. The objective of this study was to characterize the audiologic phenotype of Norrie disease for comparison with the pathophysiologic mechanism., Methods: The design combined two series of clinical audiologic evaluations, with special attention to speech intelligibility., Results: The audiologic results for 12 affected individuals and 10 carriers show that patients with Norrie disease retain high speech intelligibility scores even when the threshold loss is severe., Conclusions: The cochlear mechanism-- failure of the stria vascularis-- accounts for some of the higher values in the wide distribution of speech scores in cases with similar pure tone audiograms.

Thripinema fuscum is a natural enemy of Frankliniella fusca in peanut. Laboratory experiments were conducted to determine the reproductive biology of T. fuscum as affected by gender and stage of development of the host and to determine the effects of parasitism on host longevity, fecundity, and mortality. The adult females of F. fusca were the most readily parasitized (P < 0.001) in the laboratory experiments followed by the second instars, the first instars, and the adult males. One generation of T. fuscum developed within the parasitized larvae and adults, with the males and females emerging only during the adult stage of the host. Parasitism did not cause mortality of the host. Parasitism affected male longevity (P < 0.001) but not female longevity. The adult female thrips that were parasitized as first or second instars did not lay eggs, and the adult females stopped laying eggs within 3 days of being parasitized. The female-to-male sex ratio of T. fuscum emerging from parasitized male and female F. fusca was 22 and 18 to 1, respectively. More T. fuscum emerged from female hosts than from male hosts (P < 0.001). More emerged from hosts parasitized as larvae compared with hosts parasitized as adults (P < 0.05). The intrinsic capacity of increase of T. fuscum ranged between 0.29 and 0.37 when parasitizing the adult males and females and between 0.18 and 0.21 when parasitizing the larval males and females. Percent parasitism of F. fusca was estimated in peanut fields. The flowers were the primary site for aggregation of the adults of F. fusca and for the free-living females of T. fuscum to parasitize new hosts. As under laboratory conditions, field parasitism of adult males was less than parasitism of adult females in 2001 and 2002 (P < 0.01 and 0.001, respectively). Our study indicates that T. fuscum is a potential biological control agent capable of suppressing F. fusca populations in peanut.

Over the last quarter century, scientific advances have created new tools and technologies to improve the safety and efficacy of vaccines. These improvements are spurred by social and commercial needs to enhance existing vaccines or to create new ones against an expanding spectrum of diseases. Vaccines based on live, attenuated, pathogenic bacteria were originally developed to prevent infection by homologous pathogens. More recently, strategies have been developed to use these bacterial vaccines as vectors to deliver a variety of protective, vaccine antigens via the mucosal route. These approaches are being developed to protect not only against heterologous microbial infections, but also against non-traditional threats such as biowarfare and cancer. These strategies and their application to the recent development of delivery systems for use in humans will be discussed.

The demand for new and improved vaccines against human diseases has continued unabated over the past century. While the need continues for traditional vaccines in areas such as infectious diseases, there is an increasing demand for new therapies in nontraditional areas, such as cancer treatment, bioterrorism and food safety. Prompted by these changes, there has been a renewed interest in the application and development of live, attenuated bacteria expressing foreign antigens as vaccines. The application of bacterial vector vaccines to human maladies has been studied most extensively in attenuted strains of Salmonella. Live, attenuated strains of Shigella, Listeria monocytogenes, Mycobacterium bovis-BCG and Vibrio cholerae provide unique alternatives in terms of antigen delivery and immune presentation, however and also show promise as potentially useful bacterial vectors.

The acaricidal mycopathogen Hirsutella thompsonii has been found to secrete metabolites that are active against female Tetranychus urticae. Specifically, the rose-colored exudate produced on sporulating cultures of Mexican HtM120I strain sterilized female spider mites in a dose-dependent fashion. Topical application of the exudate resulted in a 100% reduction in mite fecundity over the initial six days of experimentation. Depending upon the exudate dosage, mites partially recovered within 3 and 6 d post-treatment and produced a limited number of eggs. The spider mite active HtM120I exudate contained less detectable HtA toxin than the HtM120I broth filtrate, and it was innocuous when injected into the greater wax moth Galleria mellonella L. larvae. Broth filtrates of HtM120I cultures, although toxic to assayed G. mellonella larvae, did not inhibit mite oviposition to the degree or duration of the exudate preparations. These findings suggest that the factor responsible for suppressing oviposition in female spider mites is linked to the sporulation process and is distinct from the well-characterized HtA produced by vegetative cells.

Tuberous sclerosis complex (TSC) is a familial hamartoma syndrome in which renal involvement is common and, at times, life threatening. We have investigated the potential effect of a non-TSC gene on renal disease in a cohort of 172 TSC patients with TSC2 mutations. Patients were genotyped for an interferon-gamma (IFN-gamma) microsatellite polymorphism, within intron 1, for which one common allele (allele 2, with 12 CA repeats) has been shown to have a higher expression of IFN-gamma. A chi(2) analysis was used to examine the association between IFN-gamma allele 2 and the development of kidney angiomyolipomas (KAMLs) in this TSC2 cohort. Because of the age-dependent development of KAMLs in TSC, we initially focused on the 127 patients who were >5 years old. Additional subgroup analyses were done to investigate the influence of age and gender. The transmission/disequilibrium test (TDT) was also performed in a subset of this cohort (46 probands) for whom parent and/or sibling samples were available for analysis. Both chi(2) analysis and TDT suggested an association between IFN-gamma allele 2 and the absence of KAMLs in patients who have known TSC2 mutations. Among the 127 patients who were >5 years old, KAMLs were present in 95 (75%) and were absent in 32 (25%). In the group with KAML present, the frequency of IFN-gamma allele 2 was 56%; in the group with KAML absent, the frequency of IFN-gamma allele 2 was significantly higher, at 78% (P=.02, by chi(2) analysis). The family-based TDT analysis gave similar results, with a TDT statistic (TDT chi2=5.45) corresponding to a P value of.02. Subgroup analyses show that both age and gender may influence the impact of this association. Although these results should be replicated in other populations with TSC, the present study suggests that modifier genes play a role in the variable expression of TSC and also suggests a potential therapy for KAMLs in patients with TSC.

Objectives: (a) To compare the magnitude of gluteus medius and tensor fascia lata activation between a group of subjects with clinical unilateral hip osteoarthritis and a group of healthy older adults. (b) To compare the magnitude of activation of the gluteus medius and tensor fascia lata between sides in a group of subjects with clinical unilateral hip osteoarthritis and a group of healthy older adults., Methods: 19 subjects with clinical unilateral hip osteoarthritis and 19 healthy controls were investigated. The subjects performed a stepping task during which recordings were obtained using surface electromyograms from the hip abductors, and kinetic data were obtained from a dual force platform., Results: Subjects with clinical hip osteoarthritis had higher gluteus medius activation than the healthy older adults (p=0.037). In addition, there were no differences in the magnitude of gluteus medius activation between the sides (p=0.733). There was no difference in the force platform data between the groups (p=0.078)., Conclusions: The increased magnitude of gluteus medius activation in the group with hip osteoarthritis is evidence of a muscular dysfunction associated with hip disease. This has implications for the progressive nature of the disease and for its conservative management.

There is an expanding understanding of primary genetic oxidative-phosphorylation disorders and the recognition of new multi-system clinical phenotypes in the energy metabolism diseases. Although initially recognized in association with mitochondrial DNA mutations, there is progress in the more laborious identification of nuclear DNA encoded genes relevant to mitochondrial structure and function. More pathogenic mitochondrial DNA and nuclear DNA mutations have been identified. Diagnosis of these disorders is often difficult and relies on a concurrence of findings, including recognition of a variety of clinical signs and symptoms, biochemical marker screening, electron transport chain enzyme measurements, and mitochondrial DNA or nuclear DNA mutation assay of genes relevant to mitochondrial structure, function or adenosine triphosphate metabolic pathways. Clinical diagnostic assessment now can be augmented by physiologic imaging techniques, including nuclear magnetic resonance spectroscopy and positron emission tomography. These capabilities should be increasingly helpful for studies of clinical progression and therapeutic intervention. Biologic studies, in families and patients, are beginning to address the factors of mitochondrial replication and segregation that underlie cellular/tissue heteroplasmy and clinical variability. Most epigenetic factors affecting organ-specific and phenotypic variability, however, remain to be elaborated.

Tuberous sclerosis complex (TSC), an autosomal dominant disease caused by mutations in either TSC1 or TSC2, is characterized by the development of hamartomas in a variety of organs. Concordant with the tumor-suppressor model, loss of heterozygosity (LOH) is known to occur in these hamartomas at loci of both TSC1 and TSC2. LOH has been documented in renal angiomyolipomas (AMLs), but loss of the wild-type allele in cortical tubers appears to be very uncommon. Analysis of second, somatic events in tumors for which the status of both TSC1 and TSC2 is known is essential for exploration of the pathogenesis of TSC-lesion development. We analyzed 24 hamartomas from 10 patients for second-hit mutations, by several methods, including LOH, scanning of all exons of both TSC1 and TSC2, promoter methylation of TSC2, and clonality analysis. Our results document loss of the wild-type allele in six of seven AMLs, without evidence of the inactivation of the second allele in many of the other lesions, including tumors that appear to be clonally derived. Laser-capture microdissection further demonstrated loss of the second allele in all three cellular components of an AML. This study thus provides evidence that, in both TSC1 and TSC2, somatic mutations resulting in the loss of wild-type alleles may not be necessary in some tumor types-and that other mechanisms may contribute to tumorigenesis in this setting.

Objective: To compare the nicotine content of 12 unfiltered brands of bidi cigarettes (hand rolled cigarettes imported from India) with 8 popular brands of filtered and unfiltered US and conventional cigarettes from India., Main Outcome Measures: Identical laboratory procedures were used to determine nicotine content (in duplicate) and physical characteristics., Results: The nicotine concentration in the tobacco of bidi cigarettes (21.2 mg/g) was significantly greater than the tobacco from the commercial filtered (16.3 mg/g) and unfiltered cigarettes (13.5 mg/g)., Conclusions: Bidi cigarettes contain higher concentrations of nicotine than conventional cigarettes. Therefore, it is logical to presume that bidi smokers are at risk of becoming nicotine dependent. These findings belief a popular belief among US teens that bidis are a safe alternative to commercial cigarettes.

Objective: To compare the AutoCyte SCREEN (AutoCyte, Burlington, North Carolina, U.S.A.) system with manual screening by experienced cytotechnologists using thin-layer preparations that had been previously extensively studied and their cytologic abnormalities well defined., Study Design: AutoCyte PREP (AutoCyte) samples prepared for a previous split-sample study comparing thin-layer preparations to conventional smears were used. These 1,992 AutoCyte PREP samples were in a cohort the abnormal findings of which had been confirmed via independent review by two sets of pathologists. For the current study, these samples were remasked and evaluated by the AutoCyte SCREEN system in a clinical laboratory. The instrument scanned each slide and selected six overview fields and 120 single objects for storage and display. The computer classified each slide in one of the following categories: abnormal, uncertain, normal or unsatisfactory. Independently for each case, a cytotechnologist evaluated the six fields and 120 objects selected by the instrument as abnormal, normal or unsatisfactory. For those cases classified as uncertain by AutoCyte, the technologist then reexamined the cellular displays and entered a consensus classification. These results were then compared to those of an independent review by cytotechnologists of the identical set of slides using routine manual screening., Results: The AutoCyte SCREEN selected 35% of slides for manual review. Technologist and computer rendered equivalent classifications in 79%. Of the total slides screened by the AutoCyte SCREEN, 57% were classified as "uncertain," and 88% of these were subsequently classified as normal by consensus. Using the well-defined abnormal values of the cellular sample as a basis for calculation, the AutoCyte SCREEN-assisted practice had a diagnostic sensitivity of 85% and diagnostic specificity of 97.6%. Comparable values for manual screening of the identical cellular sample were a diagnostic sensitivity of 80% and specificity of 97.4%., Conclusion: The AutoCyte SCREEN achieves comparable or greater sensitivity in detecting cervical abnormalities in comparison with manual screening. When combined with the substantial advantage of thin-layer preparations over conventional smears, the AutoCyte SCREEN provides a screening system of superior sensitivity over conventionally prepared and examined cervical smears.

Hip osteoarthritis (OA) can be the result of a multitude of causes. There is some evidence to suggest that a dysfunction in the neuromuscular system may contribute to the development of hip OA. This article reviews the forces and loads at the hip joint and the different presentations of hip OA in order to present a hypothesis that the neuromuscular system can play a role in the development of hip OA. Changes in the neuromuscular system include alterations in gait patterns and changes in muscular synergies. An improved understanding of the role these factors may play in the development of hip OA provides a perspective to explore treatment rationales and to provide more effective conservative management.

Brunner et al. [1993: Am J Hum Genet 52: 1032-1039; 1993: Science 262:578-580] described males with an MAO-A deficiency state resulting from a premature stop codon in the coding region of the MAOA gene. This deficiency state was associated with abnormal levels of amines and amine metabolites in urine and plasma of affected males, as well as low normal intelligence and apparent difficulty in impulse control, including inappropriate sexual behavior. In the present study, disruption of the MAOA gene was evaluated in males with mental retardation with and without a history of sexually deviant behavior, as well as normal controls, healthy males, and patients with other diseases (Parkinson disease, Lesch-Nyhan syndrome). When available, plasma samples were evaluated first for levels of 3-methoxy, 4-hydroxyphenolglycol (MHPG), a metabolite of norepinephrine which serves as the most sensitive index of MAO-A activity in humans. Blood DNA from individuals with abnormally low MHPG, and from other individuals for whom metabolite levels were not available, were screened for nucleotide variations in the coding region of the MAOA gene by single-strand conformational polymorphism (SSCP) analysis across all 15 exons and splice junctions, and by sequencing, when indicated by either altered metabolites or SSCP shifts. No evidence for mutations disrupting the MAOA gene was found in 398 samples from the target populations, including institutionalized mentally retarded males (N = 352) and males participating in a sexual disorders clinic (N = 46), as well as control groups (N = 75). These studies indicate that MAOA deficiency states are not common in humans.

Transfusion of ABO-incompatible platelets to a cardiac surgery patients resulted in acute intravascular hemolysis complicated by ventricular tachycardia. Nine similar cases with significant morbidity or mortality have been reported in similar settings. Five fatalities caused by transfusion of ABO-incompatible platelet products have been reported to the US Food and Drug Administration during the last 4 years. Non-group O patients, particularly those with small plasma volumes, receiving multiple non-group specific platelets in a short time are at risk for intravascular hemolysis caused by passively infused anti-A1 or anti-B. To prevent intravascular hemolysis in such at-risk patients, the indications for platelet transfusion must be continually assessed. If transfusion of ABO-incompatible platelet products is necessary, consideration should be given to minimizing the accompanying plasma via further concentration of the platelets or by saline washing of platelet products.

Tuberous sclerosis complex (TSC) is an autosomal dominant disorder characterized by the development of multiple hamartomas involving many organs. About two-thirds of the cases are sporadic and appear to represent new mutations. With the cloning of two causative genes, TSC1 and TSC2 it is now possible to analyze both genes in TSC patients and identify germline mutations. Here we report the mutational analysis of the entire coding region of both TSC1 and TSC2 genes in 126 unrelated TSC patients, including 40 familial and 86 sporadic cases, by single-stranded conformational polymorphism (SSCP) analysis followed by direct sequencing. Mutations were identified in a total of 74 (59%) cases, including 16 TSC1 mutations (5 sporadic and 11 familial cases) and 58 TSC2 mutations (42 sporadic and 16 familial cases). Overall, significantly more TSC2 mutations were found in our population, with a relatively equal distribution of mutations between TSC1 and TSC2 among the familial cases, but a marked underrepresentation of TSC1 mutations among the sporadic cases (P = 0.0035, Fisher's exact test). All TSC1 mutations were predicted to be protein truncating. However, in TSC2 13 missense mutations were found, five clustering in the GAP-related domain and three others occurring in exon 16. Upon comparison of clinical manifestations, including the incidence of intellectual disability, we could not find any observable differences between TSC1 and TSC2 patients. Our data help define the distribution and spectrum of mutations associated with the TSC loci and will be useful for both understanding the function of these genes as well as genetic counseling in patients with the disease., (Copyright 1999 Wiley-Liss, Inc.)

Early-onset, generalized primary torsion dystonia (PTD) is an autosomal dominantly inherited disorder, characterized by involuntary movements and abnormal postures. The majority of cases are caused by a 3-bp deletion in the DYT1 gene on chromosome 9q34 that allows for specific genetic testing. We developed a simple, reliable, and cost-effective, PCR-based screening method for this mutation. Testing results from a cohort of 550 cases, including patients with different forms of dystonia and unclassified movement disorders, revealed that 72.2% of the patients with typical early-onset generalized PTD carried the GAG deletion in the DYT1 gene. Among 300 cases with late-onset focal/segmental dystonia, only 3 patients tested positive for the GAG deletion whereas 12.8% of the patients with an unclassified movement disorder were GAG positive. Our results confirm a genotype/phenotype correlation in early-onset PTD and show that application of strict clinical criteria leads to accurate prediction of carrier status in more than two-thirds of patients with this type of dystonia. Currently, we suggest that testing be recommended in individuals with age of onset of dystonia below 30 years and/or a positive family history of early-onset PTD. Testing is not recommended in patients with onset of symptoms after 30 years or in asymptomatic individuals under the age of 18.

Glutamate and aspartate are the primary excitatory neurotransmitters in the mammalian central nervous system and have also been implicated as mediators of excitotoxic neuronal injury and death. The precise control of extracellular glutamate and aspartate is crucial to the maintenance of normal synaptic transmission and the prevention of excitotoxicity following acute insults to the brain, such as stroke or head trauma, or during the progression of neurodegenerative diseases such as amyotrophic lateral sclerosis. The removal of excitatory amino acids (EAAs) from the extracellular space is primarily mediated by a family of sodium-dependent glutamate transporters. These transporters use the sodium electrochemical gradients of the cell to actively concentrate EAAs in both neurons and glia. Five members of this transporter family have been cloned recently and include both 'glial'-specific and 'neuron'-specific subtypes. Although these subtypes share many common functional properties, there are considerable differences in developmental expression, chronic and acute regulation by cellular signaling pathways, and contribution to disease processes among the subtypes. In this review recent studies of glutamate transporter expression, regulation, function, and pathological relevance are summarized, and some of the discrepancies and unexpected results common to any rapidly progressing field are discussed.

This report details the histopathologic findings in a woman who acquired the human papillomavirus 6/11 in her late teens and developed papilloma/condyloma of the nasopharynx, oropharynx, anogenital region, urethra, and urinary bladder. General evaluations of immune function reveal no defect, and there was no evidence of HIV infection. The morphologic expression of HPV 6/11 infection appears to be completely dependent on the mucosal epithelium affected. The complete spectrum of benign and premalignant epithelial changes induced by the human papillomavirus family-papilloma, verrucae, condyloma acuminatum, epithelial hyperplasia, and dysplasia-were present in this patient with a single papillomavirus infection. We postulate that this patient has a specific immune deficiency that limits her ability to control local infection and spread of the papillomavirus.

Analysis of 122 spontaneous large and small colony mutants derived from L5178Y tk +/- mouse lymphoma cells at 28 heteromorphic microsatellite loci on chromosome 11 showed that extensive loss of heterozygosity (LOH) is common in both large colony and small colony mutants, eliminating most chromosome 11 loci as candidates for a putative growth control locus. These results, in conjunction with historical cytogenetic data, suggest that a putative growth control locus lies distal to the thymidine kinase (Tk1) gene, near the telomere. Thirty seven mutants were hybridized with a chromosome 11-specific whole chromosome painting probe for analysis of rearrangements. Generally, painting confirmed earlier observations that large colony mutants are karyotypically normal, whereas small colony mutants frequently have detectable rearrangements. A point probe distal to Tk1 revealed no evidence of chromosome breakage in small colony mutants that appeared normal on whole 11 painting and had no LOH. Therefore, the molecular difference between large and small colony mutants remains unknown. Models to explain large and small colony mutants consistent with our findings are presented, including loss of a putative growth control gene, differential mechanisms of chromosome breakage/repair and second site mutations as explanations for small colony mutants. Painting revealed translocations and aneuploidy and showed that non-disjunction was not a common explanation for complete LOH. The most common finding was that large regions of LOH do not result from deletions, demonstrating that these cells can detect recombination events as well as previously observed chromosomal rearrangements, deletions and point mutations.

Objective: To compare the diagnostic efficacy of a thin-layer technique with that of a membrane filter technique using a wide variety of fresh cytologic specimens., Study Design: Paired samples from 272 nongynecologic cytology specimens were processed for microscopy using thin-layer and membrane filter preparation techniques. Specimens included 162 body fluids and urines, 32 bronchial aspirates and 78 fine needle aspiration biopsies. The two techniques were compared for diagnostic efficacy, cell density/specimen, cell types recovered, qualitative cytomorphology, ease of interpretation and long-term stability of the final preparations., Results: Diagnoses supported by filter preparations were also supported by their matched thin-layer preparations. The same cell types were recovered by both techniques, and the slide cell density was slightly greater using membrane filters. Qualitative morphology and ease of use were superior with the thin-layer samples. After two years, all thin-layer slides were stable and readable, whereas only 43% of the membrane filter slides were usable., Conclusion: Thin-layer techniques are morphologically comparable and, in some ways, superior to membrane filter techniques for processing a wide variety of nongynecologic cytology specimens.

Objective: To determine the feasibility and utility of thin-layer cytology preparations for morphometric analysis of nuclear and cytoplasmic area in cells from nongynecologic cytology specimens., Study Design: Identical paired samples from nongynecologic specimens (bronchial aspirate, urine, pleural and peritoneal fluid) were used to prepare thin-layer preparations and corresponding traditional membrane filter preparations. The paired preparations were analyzed by static image morphometry measuring eight nuclear and cytoplasmic parameters that allowed calculation of nuclear area, cytoplasmic area and total cell size. Hepatocytes and bronchial columnar, mesothelial, squamous and transitional cells were studied, as was a single case of high grade transitional cell carcinoma. Sufficient numbers of each cell type were measured to allow statistical analysis., Results: Both thin-layer and membrane filter preparation techniques yielded individual cells suitable for morphometric analysis, and there were no consistent morphologic measurement differences between the two methods. The thin-layer preparation had the following significant technical advantages: more numerous easily measured single cells, lack of interfering background and superior specimen stability. Benign oval to round nuclei from a variety of cell types have a mean nuclear area in a narrow range from 29 to 55 microns 2. The mean nuclear area of malignant cells studied was significantly larger (78 microns 2), and there was a significant decrease in absolute cytoplasmic profile area in the malignant population studied., Conclusion: Thin-layer cytology preparations have significant advantages for morphometric studies over traditional membrane filter cytologic preparations. The morphometric measurement of nuclear area in benign and malignant cell populations has great potential as a generic screening tool for malignancy in cytologic specimens. Simultaneous measurement of cytoplasmic area adds a powerful dimension. The subsequent calculation of a true nuclear/cytoplasmic ratio may yield a sensitive and specific discriminator for detecting malignant cell populations in human nongynecologic cytology specimens.

Tuberous sclerosis complex (TSC) is a dominantly inherited multisystem disorder resulting in the development of hamartomatous growths in many organs. Genetic heterogeneity has been demonstrated linking the familial cases to either TSC1 at 9q34.3, or TSC2 at 16p13.3. About two-thirds of the TSC cases are sporadic and appear to represent new mutations. While both genes are thought to account for all familial cases, with each representing approximately 50% of the mutations, the proportion of sporadic cases with mutations in TSC1 and TSC2 is yet to be determined. We have examined the entire coding sequence of the TSC2 gene in 20 familial and 20 sporadic cases and identified a total of twenty-one mutations representing 50% and 55% of familial and sporadic cases respectively. Our rate of mutation detection is significantly higher than other published reports. Twenty out of 21 mutations are novel and include 6 missense, 6 nonsense, 5 frameshifts, 2 splice alterations, a 34 bp deletion resulting in abnormal splicing, and an 18 bp deletion which maintains the reading frame. The mutations are distributed throughout the coding sequence with no specific hot spots. There is no apparent correlation between mutation type and clinical severity of the disease. Our results document that at least 50% of sporadic cases arise from mutations in the TSC2 gene. The location of the mutations described here, particularly the missense events, should be valuable for further functional analysis of this tumor suppressor protein.

This article traces the development of pathology practice from its origins in autopsy pathology to its current practice in the United States. The American model of practice differs markedly from that in continental European countries because of the extensive incorporation of "Clinical Pathology" with the traditional disciplines of anatomic pathology under the auspices of the Pathology Department. "Clinical Pathology" as it is now defined includes the laboratory testing disciplines of Chemistry, Hematology, Immunology, Medical Microbiology, and Transfusion Medicine. The increasing importance of computers and information management, DNA diagnostic techniques, and the multiple roles of the pathologist as a researcher and consultant in pathology practice in the United States is discussed.

Lack of monoamine oxidase A (MAO-A) due to either Xp chromosomal deletions or alterations in the coding sequence of the gene for this enzyme are associated with marked changes in monoamine metabolism and appear to be associated with variable cognitive deficits and behavioral changes in humans and in transgenic mice. In mice, some of the most marked behavioral changes are ameliorated by pharmacologically-induced reductions in serotonin synthesis during early development, raising the question of possible therapeutic interventions in humans with MAO deficiency states. At the present time, only one multi-generational family and a few other individuals with marked MAO-A deficiency states have been identified and studied in detail. Although MAO deficiency states associated with Xp chromosomal deletions were identified by distinct symptoms (including blindness in infancy) produced by the contiguous Norrie disease gene, the primarily behavioral phenotype of individuals with the MAO mutation is less obvious. This paper reports a sequential research design and preliminary results from screening several hundred volunteers in the general population and from putative high-risk groups for possible MAO deficiency states. These preliminary results suggest that marked MAO deficiency states are very rare.

Objective: To assess the efficacy of 10% formalin perfusion fixation as a method of rapid fixation to examine the human brain immediately following autopsy., Design: Compare the histology and immunohistochemistry from human brains in which one hemisphere undergoes perfusion fixation using 10% buffered formalin, and the contralateral nonperfused hemisphere undergoes standard 14-day immersion fixation in 8 L of 10% buffered formalin., Setting: Autopsy material in a general medical-surgical university hospital., Participants: Pathologists, neuropathologists, resident pathologists, and pathology assistants., Intervention: Immediately following brain removal, a single hemisphere was perfused with 1 L 10% buffered formalin over a 15- to 20-minute period. The contralateral nonperfused hemisphere served as a control, undergoing standard immersion fixation for 2 weeks in 10% formalin. The perfusion-fixation hemisphere was immediately available for neuropathologic examination, and histologic sections of the brain were processed immediately with the other necropsy tissue sections. This allows completion of a final autopsy neuropathology report within 3 to 5 days in concert with the systemic section of the report., Main Outcome Measure: Perfusion-fixation brain sections were compared with immersion-fixation brain sections from the same brain. The effects on hematoxylin-eosin, Bielschowsky's silver, and immunohistochemical staining were evaluated by an experienced neuropathologist and a general pathologist with no prior knowledge of the fixation technique., Results: Perfusion fixation revealed equal and occasionally superior histologic sections compared with traditional immersion fixation in terms of (1) technical preparation of section, (2) quality and intensity of staining with both hematoxylin-eosin and silver, and (3) immunoreactivity localization with a variety of immunohistochemical reactions., Conclusions: Immediate perfusion of the brain is an easily performed fixation technique that yields comparable or superior fixation to prolonged immersion fixation and allows an immediate complete neuropathologic examination and report within 3 to 5 days of performance of the autopsy.

One hundred seventy-six United States and Canadian pathology residency programs were surveyed to assess the status of management training for residents. Specific questions in the survey concerned approaches to management, instructional methods used, topics covered, and length of management rotations. Eighty-four programs (48%) responded to the survey. This group represented a cross section of all programs as to location, size, and type of program. Of the 84 programs, 81 (96%) offered management training, while 54 programs (64%) offered a management course. Management training usually occurred in combination with other rotations, most frequently clinical pathology subspecialties. A dedicated management rotation was reported in 19 programs (23%). The most common method of instruction was apprenticeship/mentor followed by didactic lecture. A majority of programs used multiple instructional methods. The five most frequently covered topics were budgets, personnel issues, quality assurance, utilization, and instrument evaluation, but a wide variety of topics were included in management curricula surveyed. There was an emphasis on clinical laboratory administration. Nonpathologist instructors, such as medical technologists, hospital administrators, and business executives, were used for formal didactic exercises, while teaching by pathologists occurred predominantly by mentoring during clinical pathology rotations.

Leadership and management skills are increasingly critical to the success of pathologists in and outside of academic centers. We describe a leadership and management curriculum in a combined anatomic and clinical pathology residency program. The mentor-based approach incorporates leadership and management skills throughout the residency with a dedicated 2-month rotation during the final year of training. This 2-month rotation is led by a senior faculty pathologist mentor and includes active participation in management activities, small group discussions, reading, and a management process project. The topics for the small group discussions, reading list, mentor-based activities, and completed management process projects are described. Outcomes and evaluations are reported for residents who have completed this curriculum in leadership and management.

Objectives: To show that Norrie disease can occur in a girl and to describe her ophthalmologic and genetic features., Methods: Amplification of DNA polymerase chain reaction and sequencing of asymmetric polymerase chain reaction for exon 3 were performed on the blood specimen obtained from a girl born with bilateral retinal detachments., Patient: A female child with bilateral retinal detachment who had 2 uncles in whom Norrie disease had already been diagnosed., Results: The child had a mutation in the third exon (T776-->A; Ile 123-->Asn) identical to the mutation found in her uncles., Conclusions: Norrie disease can occur in girls. The most likely explanation is nonrandom or unfavorable X inactivation, although timing of development of the peripheral retina and its blood supply could render it vulnerable to effects of the mutant allele at a critical developmental phase.

A large Costa Rican kindred has been identified with 15 males affected with congenital blindness, progressive bearing loss, and venous insufficiency. Due to ophthalmological and audio-otological findings, including bilateral retinal dysplasia and detachment, progressive bilateral sensorineural hearing loss, and an X-linked pattern of inheritance, a tentative diagnosis of Norrie disease was considered. However, venous insufficiency is a clinical finding not reportedly associated with Norrie disease. Genetic linkage analysis using microsatellite repeat markers demonstrated linkage to Xp11.23-11.4 (z = 2.723 at theta = 0.0). A candidate gene approach using the Norrie disease gene (NDP), which maps to Xp11.3, revealed a point mutation in the third exon resulting in substitution of phenylalanine for leucine at position 61. The precise function of the gene product, norrin, has yet to be elucidated; however, it has been postulated to be involved in the regulation of neural cell differentiation and proliferation, although hypotheses have been considered for its role in vascular development in the eye. The finding of a mutation in NDP in association with peripheral vascular disease may provide valuable insight into the potential role of this gene in cellular processes.

Background: The Norrie disease (ND) gene (Xp11.3) (McKusick 310600) consists of one untranslated exon and two exons partially translated as the Norrie disease protein (Norrin). Norrin has sequence homology and computer-predicted tertiary structure of a growth factor containing a cystine knot motif, which affects endothelial cell migration and proliferation. Norrie disease (congenital retinal detachment), X-linked primary retinal dysplasia (congenital retinal fold), and X-linked exudative vitreoretinopathy (congenital macular ectopia) are allelic disorders., Methods: Blood was drawn for genetic studies from members of two families to test for ND gene mutations. Sixteen unaffected family members were examined ophthalmologically. If any retinal abnormality were identified, fundus photography and fluorescein angiography was performed., Results: Family A had ND (R109stp), and family B had X-linked exudative vitreoretinopathy (R121L). The retinas of 11 offspring of carrier females were examined: three of seven carrier females, three of three otherwise healthy females, and one of one otherwise healthy male had peripheral inner retinal vascular abnormalities. The retinas of five offspring of affected males were examined: none of three carrier females and none of two otherwise healthy males had this peripheral retinal finding., Conclusions: Peripheral inner retinal vascular abnormalities similar to regressed retinopathy of prematurity were identified in seven offspring of carriers of ND gene mutations in two families. These ophthalmologic findings, especially in four genetically healthy offspring, strongly support the hypothesis that abnormal Norrin may have an adverse transplacental (environmental) effect on normal inner retinal vasculogenesis.

Objective: To develop a system for enhancing the performance and reporting of autopsies in an effective and clinically useful manner., Design: Twelve steps were defined as essential for the completion of the autopsy. Each step of the process was evaluated for usefulness and effectiveness., Setting: Autopsies performed in a university hospital from 1992 through 1994., Participants: Pathology residents and staff, clinicians, and clinical team house staff., Intervention: Participants followed the 12-step process, with emphasis on involving the clinical team in the interview, prosection, and final rounds. The final rounds conference was designated a working conference, where the perfused-fixed brain was cut, histologic sections of the case were submitted, and the provisional diagnosis was written with the clinicians. A next-day microscopic slide review session was scheduled to "sign out" the case. Establishing a philosophy of status equal to all other department functions facilitated implementation., Main Outcome Measure: All autopsies performed for a period of 3 years (2 retrospective and 1 prospective) were included., Results: The autopsy completion time was reduced from a mean of 57 days (range 7 to 174) in 1992 to 4.8 days (range 1 to 16) in 1994., Conclusion: The autopsy completion time was reduced, increasing its usefulness for teaching and quality assurance. Relationships with the clinical staff were enhanced with consultation-style final reports. Enthusiasm for, and satisfaction with, the new process was expressed by clinicians, pathology staff, residents, and technical support staff.

We have established a method for preparing cDNA sublibraries enriched in sequences from specific chromosome regions, called selection of hybrids by affinity capture (SHAC). This procedure can be described in two stages. In the first stage, a particular chromosome region, in this study mouse chromosome 11, was microdissected, followed by PCR amplification with a universal degenerate primer. This material is referred to as the "target" DNA. In the second stage, a mouse liver cDNA library with unique linker-adapter ends, referred to as the "source" cDNA, was hybridized to the biotin-labeled target DNA prepared during the first stage. The resulting DNA duplexes were captured by streptavidin-coated magnetic beads. The cDNAs were released from their biotin-labeled target homologs by alkaline denaturation and recovered by PCR amplification. These cDNAs were referred to as the SHACcDNAs. Specificity of the SHACcDNA to chromosome 11 was verified by FISH analysis. To examine representation of the SHACcDNA, we confirmed the presence of seven genes or single-copy DNA segments known to be localized on mouse chromosome 11, using a dot blot assay. In addition, a second round of SHAC was performed to achieve even higher specificity for the resulting chromosome 11 SHACcDNA. The SHAC technology should facilitate construction of cytogenetically defined cDNA libraries and should assist in the fields of gene discovery and genome mapping.

Quality assurance (QA) in surgical pathology has focused primarily on retrospective audits of randomly selected cases. The authors describe an effective method of prospective audit for a selected class of surgical specimens--diagnostic biopsies--and document the benefits, additional staff time required and impact on turnaround time. Additionally, these results were compared with a retrospective review. During a 6-month period, all diagnostic surgical pathology biopsies (n = 2,694, 55% of all cases) were reviewed by a second pathologist before release of the final report. Errors detected were subdivided into four categories: (1) major: errors in diagnosis that could directly affect patient care; (2) diagnostic discrepancies: errors in diagnosis that should not affect patient care; (3) minor: correct diagnosis rendered, but report correction required to add supportive information; (4) clerical: typographical and grammatical errors. Thirty-two major errors were found, involving 1.2% of cases reviewed. This manner of review caused an increase in overall turnaround time from 1.62 days to 1.79 days, and an increase in turnaround time for diagnostic biopsies from 1.44 days to 1.50 days. Time spent in performing prospective peer review averaged 4 hours per day. For comparison, results were included from a retrospective review performed on 480 of the 5,556 cases accessioned in a 6-month period before the institution of prospective quality assurance. This retrospective review revealed eight major errors (1.7%). In conclusion, the prospective peer review of diagnostic biopsies yields sufficient benefits in increased accuracy of diagnostic reports to justify the slight increase in additional work by pathologists.

The effect of dissolved oxygen on citric acid production and oxygen uptake by Candida lipolytica Y 1095 was evaluated in cell recycle and fed-batch fermentation systems. The maximum observed volumetric productivity, which occurred at a dilution rate of 0.06 h(-1), a dissolved oxygen concentration of 80%, and a biomass concentration of 5% w/v, in the cell recycle system, was 1.32 g citric acid/L . h. At these same conditions, the citric acid yield was 0.65 g/g and the specific citric acid productivity was 24.9 mg citric acid/g cell . h. In the cell recycle system, citric acid yields ranged from 0.45 to 0.72 g/g. Both the volumetric and specific citric acid productivities were dependent on the dilution rate and the concentration of dissolved oxygen in the fermentor. Similar productivities (1.29 g citric acid/L . h) were obtained in the fed-batch system operated at a cycle time of 36 h, a dissolved oxygen concentration of 80%, and 60 g total biomass. Citric acid yields in the fed-batch fermentor were consistently lower than those obtained in the cell recycle system and ranged from 0.40 to 0.59 g/g. Although citric acid yields in the fed-batch fermentor were lower than those obtained in the cell recycle system, higher citric:isocitric acid ratios were obtained in the fed-batch fermentor. As in the cell recycle system, both the volumetric and specific citric acid productivities in the fed-batch fermentor were dependent on the cycle time and dissolved oxygen concentration. (c) 1995 John Wiley & Sons, Inc.

Fucosidosis is a rare lysosomal storage disorder with the clinical features of mental retardation, cardiomegaly, dysostosis multiplex, progressive neurologic deterioration, and early death. The neuroradiologic findings in two patients are reported, and include abnormalities within the globus pallidus (both patients) and periventricular white matter (one patient).

Measurements of uranium/thorium and samarium/neodymium isotopes and concentrations in a suite of Hawaiian basalts show that uranium/thorium fractionation varies systematically with samarium/neodymium fractionation and major-element composition; these correlations can be understood in terms of simple batch melting models with a garnet-bearing peridotite magma source and melt fractions of 0.25 to 6.5 percent. Midocean ridge basalts shows a systematic but much different relation between uranium/thorium fractionation and samarium/neodymium fractionation, which, although broadly consistent with melting of a garnet-bearing peridotite source, requires a more complex melting model.

Baculovirus recombinants that express the Rickettsia rickettsii rOmpA protein were constructed. Monoclonal antibodies (mAbs) against the rOmpA protein reacted with recombinant-infected Spodoptera frugiperda (Sf9) cells in indirect immunofluorescence assays. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting of infected Sf9 cell lysates with a mAb against rOmpA showed that the recombinant-expressed rOmpA protein migrated slightly below rOmpA extracted from R. rickettsii. Guinea-pigs immunized with lysates of recombinant-infected Sf9 cells developed antibodies reactive with R. rickettsii and were protected against challenge, indicating that the baculovirus-expressed rOmpA protein could be useful in subunit vaccines and for studies of the immune response to R. rickettsii infection.

We report our experience to date in mutation identification in the Norrie disease (ND) gene. We carried out mutational analysis in 26 kindreds in an attempt to identify regions presumed critical to protein function and potentially correlated with generation of the disease phenotype. All coding exons, as well as noncoding regions of exons 1 and 2, 636 nucleotides in the noncoding region of exon 3, and 197 nucleotides of 5' flanking sequence, were analyzed for single-strand conformation polymorphisms (SSCP) by polymerase chain reaction (PCR) amplification of genomic DNA. DNA fragments that showed altered SSCP band mobilities were sequenced to locate the specific mutations. In addition to three previously described submicroscopic deletions encompassing the entire ND gene, we have now identified 6 intragenic deletions, 8 missense (seven point mutations, one 9-bp deletion), 6 nonsense (three point mutations, three single bp deletions/frameshift) and one 10-bp insertion, creating an expanded repeat in the 5' noncoding region of exon 1. Thus, mutations have been identified in a total of 24 of 26 (92%) of the kindreds we have studied to date. With the exception of two different mutations, each found in two apparently unrelated kindreds, these mutations are unique and expand the genotype database. Localization of the majority of point mutations at or near cysteine residues, potentially critical in protein tertiary structure, supports a previous protein model for norrin as member of a cystine knot growth factor family (Meitinger et al., 1993). Genotype-phenotype correlations were not evident with the limited clinical data available, except in the cases of larger submicroscopic deletions associated with a more severe neurologic syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)

The rates of oxygen uptake and oxygen transfer during cell growth and citric acid production by Candida lipolytica Y 1095 were determined. The maximum cell growth rate, 1.43 g cell/L x h, and volumetric oxygen uptake rate, 343 mg O(2)/L x h, occurred approximately 21 to 22 h after inoculation. At the time of maximum oxygen uptake, the biomass concentration was 1.3% w/v and the specific oxygen uptake rate was slightly greater than 26 mg O(2)/g cell x h. The specific oxygen uptake rate decreased to approximately 3 mg O(2)/g cell x h by the end of the growth phase.During citric acid production, as the concentration of dissolved oxygen was increased from 20% to 80% saturation, the specific oxygen uptake and specific citric acid productivity (mg citric acid/g cell x h) increased by 160% and 71%, respectively, at a biomass concentration of 3% w/v. At a biomass concentration of 5% w/v, the specific oxygen uptake and specific citric acid productivity increased by 230% and 82%, respectively, over the same range of dissolved oxygen concentrations.The effect of dissolved oxygen on citric acid yields and productivities was also determined. Citric acid yields appeared to be independent of dissolved oxygen concentration during the initial production phase; however, volumetric productivity (g citric acid/L x h) increased sharply with an increase in dissolved oxygen. During the second or subsequent production phase, citric acid yields increased by approximately 50%, but productivities decreased by roughly the same percentage due to a loss of cell viability under prolonged nitrogen-deficient conditions.