Secondary follicle growth and oocyte maturation during encapsulated three-dimensional culture in rhesus monkeys: effects of gonadotrophins, oxygen and fetuin.

BACKGROUND: An alginate-based matrix supports the three-dimensional (3D) architecture of non-human primate follicles and, in the presence of FSH, permits the in vitro development of pre-antral follicles to the small antral stage, including the production of ovarian steroids and paracrine factors. The current study investigated the ability of gonadotrophins, fetuin and oxygen (O2) to improve primate follicle growth and oocyte maturation in vitro. METHODS: Macaque secondary follicles were isolated from the early follicular phase ovaries, encapsulated in a sodium alginate matrix and cultured individually for 40 days in supplemented medium. The effects of recombinant human (rh) FSH (15, 3 and 0.3 ng/ml for high, medium and low FSH, respectively), bovine fetuin (1 or 0 mg/ml) and O2 (5 or 20% v/v) were examined. Half of the follicles in each culture condition received rhLH on Day 30-40. Follicles that reached antral stage were treated with rh chorionic gonadotrophin for 34 h to initiate oocyte meiotic maturation. Media were analyzed for ovarian steroids and anti-müllerian hormone (AMH). RESULTS: Improved culture conditions supported non-human primate, secondary follicle growth to the antral stage and, for the first time, promoted oocyte maturation to the MII stage. In the presence of fetuin at 5% O2, follicles had the highest survival rate if cultured with high or medium FSH, whereas follicles grew to larger diameters at Week 5 in low FSH. Oocyte health and maturation were promoted under 5% O2. High FSH stimulated steroid production by growing follicles, and steroidogenesis by follicles cultured with low FSH was promoted by LH. AMH biosynthesis was elevated with high compared with low FSH and for longer under 5% O2 than under 20% O2. CONCLUSIONS: This encapsulated 3D culture model permits further studies on the endocrine and local factors that influence primate follicle growth and oocyte maturation, with relevance to enhancing fertility preservation options in women.

Executive functioning in Alzheimer's disease and vascular dementia.

OBJECTIVE: To compare performance of patients with mild-moderate Alzheimer's disease (AD) and vascular dementia (VaD) on tests of executive functioning and working memory. METHODS: Patients with AD (n = 76) and VaD (n = 46) were recruited from a memory clinic along with dementia free participants (n = 28). They underwent specific tests of working memory from the Cognitive Drug Research (CDR) battery and pen and paper tests of executive function including CLOX 1 & 2, EXIT25 and a test of verbal fluency (COWAT). All patients had a CT brain scan which was independently scored for white matter change/ischaemia. RESULTS: The AD and VaD groups were significantly impaired on all measures of working memory and executive functioning compared to the disease free group. There were no significant differences between the AD and VaD groups on any measure. Z-scores confirmed the pattern of impairment in executive functioning and working memory was largely equivalent in both patient groups. Small to moderate correlations were seen between the MMSE and the neurocognitive scores in both patient groups and the pattern of correlations was also very similar in both patient groups. CONCLUSIONS: This study demonstrates sizeable executive functioning and working memory impairments in patients with mild-moderate AD and VaD but no significant differences between the disease groups.

Effects of amisulpride, risperidone and chlorpromazine on auditory and visual latent inhibition, prepulse inhibition, executive function and eye movements in healthy volunteers.

In view of the evidence that cognitive deficits in schizophrenia are critically important for long-term outcome, it is essential to establish the effects that the various antipsychotic compounds have on cognition, particularly second-generation drugs. This parallel group, placebo-controlled study aimed to compare the effects in healthy volunteers (n = 128) of acute doses of the atypical antipsychotics amisulpride (300 mg) and risperidone (3 mg) to those of chlorpromazine (100 mg) on tests thought relevant to the schizophrenic process: auditory and visual latent inhibition, prepulse inhibition of the acoustic startle response, executive function and eye movements. The drugs tested were not found to affect auditory latent inhibition, prepulse inhibition or executive functioning as measured by the Cambridge Neuropsychological Test Battery and the FAS test of verbal fluency. However, risperidone disrupted and amisulpride showed a trend to disrupt visual latent inhibition. Although amisulpride did not affect eye movements, both risperidone and chlorpromazine decreased peak saccadic velocity and increased antisaccade error rates, which, in the risperidone group, correlated with drug-induced akathisia. It was concluded that single doses of these drugs appear to have little effect on cognition, but may affect eye movement parameters in accordance with the amount of sedation and akathisia they produce. The effect risperidone had on latent inhibition is likely to relate to its serotonergic properties. Furthermore, as the trend for disrupted visual latent inhibition following amisulpride was similar in nature to that which would be expected with amphetamine, it was concluded that its behaviour in this model is consistent with its preferential presynaptic dopamine antagonistic activity in low dose and its efficacy in the negative symptoms of schizophrenia.

Oocyte-somatic cell communication.

The physical interface between the female germ line and enveloping somatic cells is dynamically modified throughout the course of folliculogenesis. How selective pathways for communication between the oocyte and granulosa cell are established and regulated remains to be determined, but insights into the structural basis for this communication are emerging. This review summarizes the available evidence that supports the notion that the integration of oogenesis with folliculogenesis is achieved by regulated cell interactions between oocytes and granulosa cells.

Polymorphic internal transcribed spacer region 1 DNA sequences identify medically important yeasts.

Species-specific polymorphisms in the noncoding internal transcribed spacer 2 (ITS2) region of the rRNA operon provide accurate identification of clinically significant yeasts. In this study, we tested the hypothesis that ITS1 noncoding regions contain diagnostically useful alleles. The length of ITS1 region PCR products amplified from 40 species (106 clinical strains, 5 reference strains, and 30 type strains) was rapidly determined with single-base precision by automated capillary electrophoresis. Polymorphisms in the PCR product length permitted 19 species to be distinguished by ITS1 alone, compared with 16 species distinguished by using only ITS2. However, combination of both ITS alleles permitted identification of 30 species (98% of clinical isolates). The remaining 10 species with PCR products of similar sizes contained unique ITS alleles distinguishable by restriction enzyme analysis. DNA sequence analysis of amplified ITS1 region DNA from 79 isolates revealed species-specific ITS1 alleles for each of the 40 pathogenic species examined. This provided identification of unusual clinical isolates, and 53 diagnostic ITS1 sequences were deposited in GenBank. Phylogenetic analyses based on ITS sequences showed a similar overall topology to 26S rRNA gene-based trees. However, different species with identical 26S sequences contained distinct ITS alleles that provided species identification with strong statistical support. Together, these data indicate that the analysis of ITS polymorphisms can reliably identify 40 species of clinically significant yeasts and that the capacity for identifying potentially new pathogenic species by using this database holds significant promise.

Bacterial antigens elicit T cell responses via adaptive and transitional immune recognition.

T cells are a critical component of host immune responses against bacterial pathogens. T cell activation relies on recognition of antigen(s) derived from the bacteria, and this activation triggers potent biological effector mechanisms. Therefore, the characterization of antigens that are stimulatory for T cells provides insight into host-pathogen interactions and advances rational vaccine design. The adaptive immune response is defined by its ability to detect variable or unique single-gene products, whereas a 'transitional' immune system recognizes more conserved structures or products of multigene pathways. This transitional system functionally overlaps the canonical innate and adaptive immune responses. Antigen identification has relied upon biochemistry, genetics and expression cloning strategies. The development of computational approaches, fuelled by advances in immunology and genomic information, will facilitate the discovery of antigens and expand our understanding of both beneficial and pathological immune responses.

Diversity within reference strains of Corynebacterium matruchotii includes Corynebacterium durum and a novel organism.

Corynebacterium matruchotii has been the subject of numerous dental pathogenesis studies. The purpose of the present study was to resolve concerns about diversity within the reference strains of C. matruchotii through analysis of seven strains procured from the American Type Culture Collection (ATCC). Analysis of whole-cell fatty acid profiles with the library generation software of Microbial ID Inc. revealed that three types of organisms have been deposited in the ATCC as C. matruchotii. These three groups of organisms were also distinguishable by DNA-DNA dot blot hybridization, by sequences of two hypervariable regions of the 16S rRNA gene, and by the pyrrolidonyl arylamidase test. These studies indicate that two C. matruchotii reference strains, ATCC 33449 and ATCC 33822, are members of the recently proposed species, Corynebacterium durum. The colonial morphology and biochemical reactions of the C. durum strains are more diverse than originally reported. Strain ATCC 43833 is unique and represents a novel species. In addition to the type strain, ATCC 14266, true members of the species C. matruchotii include ATCC strains 14265, 33806, and 43832 plus two reference strains, L2 and Richardson 13, which comprise the vast majority of strains used in dental pathogenesis research with this species.

Characterization of the X-linked murine centrin Cetn2 gene.

A multi-gene family (Cetn1, Cetn2, and Cetn3) encodes the calcium-binding protein, centrin, in the mouse. This work characterizes the Cetn2 gene. Structurally, Cetn2 consists of five exons and four introns, and contains a classical TATA-less promoter. Cetn2 has two alternate transcription start sites, and a single length 3' untranslated region. Fluorescence in situ hybridization demonstrates that Cetn2 is an X-linked gene whose alleles replicate asynchronously during S-phase. Cetn2 encodes a 172 amino acid protein, with a predicted molecular mass of 19,795 Da (pI=4.71), that contains all of the defining characteristics of centrin. Northern blot analysis indicates that Cetn2 is ubiquitously expressed in the tissues of adult mice. RT-PCR shows that Cetn2 and Cetn3, but not Cetn1, are expressed in NIH 3T3 cells. Immunofluorescence microscopy demonstrates that mouse centrin 2 protein localizes to the region immediately surrounding the centrioles in the centrosome of NIH 3T3 cells.

Identification of medically important yeasts using PCR-based detection of DNA sequence polymorphisms in the internal transcribed spacer 2 region of the rRNA genes.

Identification of medically relevant yeasts can be time-consuming and inaccurate with current methods. We evaluated PCR-based detection of sequence polymorphisms in the internal transcribed spacer 2 (ITS2) region of the rRNA genes as a means of fungal identification. Clinical isolates (401), reference strains (6), and type strains (27), representing 34 species of yeasts were examined. The length of PCR-amplified ITS2 region DNA was determined with single-base precision in less than 30 min by using automated capillary electrophoresis. Unique, species-specific PCR products ranging from 237 to 429 bp were obtained from 92% of the clinical isolates. The remaining 8%, divided into groups with ITS2 regions which differed by /=99%. Seven clinical isolates contained ITS2 sequences that did not agree with their phenotypic identification, and ITS2-based phylogenetic analyses indicate the possibility of new or clinically unusual species in the Rhodotorula and Candida genera. This work establishes an initial database, validated with over 400 clinical isolates, of ITS2 length and sequence polymorphisms for 34 species of yeasts. We conclude that size and restriction analysis of PCR-amplified ITS2 region DNA is a rapid and reliable method to identify clinically significant yeasts, including potentially new or emerging pathogenic species.

Early detection of hepatitis C allograft reinfection after orthotopic liver transplantation: a molecular and histologic study.

After orthotopic liver transplantation (OLT), patients with chronic hepatitis C virus (HCV) infection show nearly universal persistence of viremia and reinfection of the liver, but identifying the point at which the liver is reinfected morphologically can be difficult. One tool that may potentially be useful to detect reinfection is reverse transcriptase-polymerase chain reaction (RT-PCR), which has proven to be highly sensitive for detecting HCV RNA in formalin-fixed paraffin-embedded liver tissue. Our purpose was to gain insight into the time frame of HCV reinfection by assaying for HCV RNA in serial posttransplant liver biopsy specimens. Our study population consisted of 14 patients who underwent liver transplantation for hepatitis C and had confirmed HCV RNA in pretransplant serum, absence of HCV RNA in donor livers, and available consecutive posttransplant liver allograft specimens. We performed RT-PCR for HCV RNA in serial posttransplant liver biopsy specimens, beginning at 1 week until at least one biopsy from each tested positive. HCV RNA was detected in liver tissue by RT-PCR in 1-week post-OLT liver samples in 6 of 14 (42.8%) patients, the earliest being 5 days post-OLT. Eventually, each of the remaining eight samples became RT-PCR positive as well; the first detections occurred in these at 3 weeks (three cases), 4 weeks (three cases), 48 weeks (one case), and 144 weeks (one case). Histologic identification of hepatitis C recurrence was relatively insensitive in relation to these molecular data. These data suggest that (1) HCV RNA reinfection is nearly universal after liver transplantation in patients with chronic hepatitis C infection, (2) molecular reinfection by HCV occurs at a variable interval post-OLT, with the majority of allograft livers reinfected as early as 1 week, and (3) morphologic features of hepatitis C are usually appreciable at the time of "molecular" recurrence.

Application of 16S rRNA gene sequencing to identify Bordetella hinzii as the causative agent of fatal septicemia.

We report on the first case of fatal septicemia caused by Bordetella hinzii. The causative organism exhibited a biochemical profile identical to that of Bordetella avium with three commercial identification systems (API 20E, API 20 NE, and Vitek GNI+ card). However, its cellular fatty acid profile was not typical for either B. avium or previously reported strains of B. hinzii. Presumptive identification of the patient's isolate was accomplished by traditional biochemical testing, and definitive identification was achieved by 16S rRNA gene sequence analysis. Phenotypic features useful in distinguishing B. hinzii from B. avium were production of alkali from malonate and resistance to several antimicrobial agents.

Centrosomes and cancer.

The centrosome functions as the major microtubule organizing center (MTOC) of the cell and as such it determines the number, polarity, and organization of interphase and mitotic microtubules. Cytoplasmic organization, cell polarity and the equal partition of chromosomes into daughter cells at the time of cell division are all dependent on the normal function of the centrosome and on its orderly duplication, once and only once, in each cell cycle. Malignant tumor cells show characteristic defects in cell and tissue architecture and in chromosome number that can be attributed to inappropriate centrosome behavior during tumor progression. In this review, we will summarize recent observations linking centrosome defects to disruption of normal cell and tissue organization and to chromosomal instability found in malignant tumors.

Endoscopic plantar fasciotomy: a multi-surgeon prospective analysis of 652 cases.

The authors have analysed 652 cases in which an endoscopic plantar fasciotomy was performed. These procedures were performed and clinically evaluated by 25 different surgeons, who completed an accredited cadaveric training symposium. In connection with each endoscopic plantar fasciotomy performed, the participating surgeons provided patient data on evaluation forms which were subsequently submitted to the authors. The results of this study demonstrate that the endoscopic plantar fasciotomy has become an established procedure with a high rate of success. Additionally, the procedure is minimally traumatic, and allows patients to resume regular activities quickly with less pain or discomfort than traditional methods of surgical management.

Endoscopic heel anatomy: analysis of 200 fresh frozen specimens.

The authors radiographed and dissected 200 fresh frozen cadaveric specimens selected randomly from the general United States population. A 21% incidence of inferior calcaneal exostosis formation was identified. Of those specimens identified as having an inferior calcaneal exostosis, there was a 52.4% incidence of heel spurs that were in the plantar fascia and a 47.6% incidence of heel spurs that were identified superior to the plantar fascia. After dissection of the specimens, the mean width and thickness of the medial, central and lateral bands of the plantar fascia, and the width of the medial and lateral subcutaneous fat were calculated. The presence of an inferior calcaneal bursa was identified in one specimen, and the presence of a heel neuroma was identified in 0 specimens of the 200 examined. The results of this study will assist the practitioner in performing the endoscopic plantar fasciotomy by providing the surgeon with quantitative averages of fascial dimensions. By knowing these fascial measurements, the practitioner will be aided intraoperatively in determining what level of fasciotomy to perform. This could help obviate some of the postoperative biomechanical sequelae that can occur with total releases, and immediate postoperative excessive ambulation by the patient. This study may help to gain insight into the true etiology of heel spur syndrome/plantar fasciitis.

Endoscopic decompression for intermetatarsal nerve entrapment--the EDIN technique: preliminary study with cadaveric specimens; early clinical results.

The authors have developed an endoscopic approach to the surgical treatment of Morton's neuroma. This technique is based on the premise that the condition is primarily a nerve entrapment disease. As with other endoscopically assisted procedures, the authors believe that this technique will be less traumatic, allowing an earlier return to normal activity, with less patient discomfort than with traditional surgical techniques.

Endoscopic plantar fasciotomy.

Endoscopic plantar fasciotomy (EPF) is an efficacious, minimally invasive procedure for the surgical intervention of heel spur syndrome/plantar fasciitis. Because of the minimal trauma involved with the technique, patients are able to return to full activity much quicker than with traditional "open" heel spur surgery techniques. The EPF procedure is not difficult to perform but requires extreme precision to ensure a successful result and to avoid iatrogenic complications. The author strongly encourages proper training with cadaveric specimens before using this technique with patients to ensure the highest standard of care. As with all surgical techniques, there are inherent complications; these complications are usually biomechanical in nature and can be reduced greatly by proper surgical techniques.

Endoscopic plantar fasciotomy: two portal endoscopic surgical techniques--clinical results of 65 procedures.

The authors have developed two endoscopic techniques that, in their opinion, are minimally traumatic and achieve superior clinical results when compared with traditional types of heel spur surgery. From the original development of the one-portal system for endoscopic plantar fasciotomy, a two-portal system has been developed (patent pending) that provides significant advantages over the one-portal system. Postoperative morbidity was decreased with both endoscopic techniques when compared with traditional types of open heel spur surgery. Both groups of patients returned to normal activity sooner than would patients who have had their heel spur syndrome/plantar fasciitis treated with open, traditional surgery.

Endoscopic plantar fasciotomy for chronic plantar fasciitis/heel spur syndrome: surgical technique--early clinical results.

A new, minimally traumatic endoscopic approach to plantar fasciotomy has been developed by the authors. This technique can be performed comfortably under a local anesthetic. Patients are immediately weightbearing and all returned to regular type shoes on the 3rd postoperative day. An earlier return to regular activity and work, with less pain and patient discomfort was found, as compared with traditional heel spur surgery techniques.

Endoscopic plantar fasciotomy: preliminary study with cadaveric specimens.

The authors have developed an endoscopic approach to a plantar fasciotomy. This technique would minimize the surgical trauma that is normally induced with a conventional type of heel spur surgery. The authors believe that this new technique will provide an earlier return to normal ambulation, less loss of work, and earlier, overall increased patient comfort.