Tiered approaches to chromatographic bioanalytical method performance evaluation: recommendation for best practices and harmonization from the Global Bioanalysis Consortium harmonization team.

The A2 harmonization team, a part of the Global Bioanalysis Consortium (GBC), focused on defining possible tiers of chromatographic-based bioanalytical method performance. The need for developing bioanalytical methods suitable for the intended use is not a new proposal and is already referenced in regulatory guidance language. However, the practical implementation of approaches that differ from the well-established full validation requirements has proven challenging. Advances in technologies, the need to progress drug development more efficiently, and emerging new drug compound classes support the use of categorized tiers of bioanalytical methods. This paper incorporated the input from an international team of experienced bioanalysts to surmise the advantages and the challenges of tiered approaches and to provide recommendations on paths forward.

Method transfer, partial validation, and cross validation: recommendations for best practices and harmonization from the global bioanalysis consortium harmonization team.

This paper presents the recommendations of the Global Bioanalytical Consortium Harmonization Team on method transfer, partial validation, and cross validation. These aspects of bioanalytical method validation, while important, have received little detailed attention in recent years. The team has attempted to define, separate, and describe these related activities, and present practical guidance in how to apply these techniques.

Evaluation of mental status HIV-infected patients: implications for treatment.

OBJECTIVES: The aim of the study was to explore factors that may influence mental health status of HIV-infected patients, and to figure out a method that could effectively classify the patients by evaluating the severity of their mental health problems. METHODS: Eighty-five patients were recruited and divided into two groups: the low risk group (LRG, n = 38) and the high risk group (HRG, n = 47). All patients finished Symptom Check-List 90 Revised (SCL-90-R) which is a multidimensional questionnaire designed to screen for a broad range of psychological problems. RESULTS: SCL-90-R scores of HRG were significantly higher than those of the general population and did not differ from those of psychosomatic outpatients. Scores of LRG were significantly lower than those of psychosomatic outpatients and did not differ from those of the general population in most subscales. HIV-infected men having sex with men and unemployed patients had much higher incidence of mental health problems. CONCLUSION: Besides undistinguishable group psychotherapy, we call for a clinical screening by psychological questionnaires at the first step, and then at-risk or high-risk patients should be given corresponding and individualized treatments. More attention and health care should be given to HIV-infected men who have sex with men and unemployed patients.

Study of T cell subsets and IL-7 protein expression in HIV-1-infected patients after 7 years HAART.

OBJECTIVE: To study the changes in T cell subsets and IL-7 in HIV-1-infected patients after seven years of highly active antiretroviral therapy (HAART). METHODS: Seventy-five individuals were included in this study (25 with effective HAART, 18 with ineffective HAART, 17 untreated HIV+ patients, and 15 volunteers in the HIV negative control group). The counts of CD4+, CD8+, CD8/CD38+, and CD8/HLADR+ T cells as well as the IL-7 protein expression was measured at 5 time points during a period of seven years in patients starting HAART (baseline) and in the HIV negative control group. The expression of CD127 on CD3+ T cells was measured by flow cytometry at a single time point (after 7 years) in patients with HAART and was compared with untreated HIV+ patients and the HIV negative control group. RESULTS: At baseline CD4+ T cell counts of HIV-1-infected patients were lower than that in the control group (p < 0.01), whereas the CD8+, CD8/HLADR+ and CD8/CD38+ T cell counts were higher than those in the control group (p < 0.01). After seven years of effective HAART, the CD4+ T cell counts had increased and the CD8+ T cell count had decreased, although not to the normal levels (p < 0.05). Both the CD8/HLADR+ and CD8/CD38+ T cell counts had gradually approached those of the control group (p > 0.05). In the ineffective HAART group, the CD8/CD38+ T cell count had not decreased significantly, and CD8/HLADR+ T cell count gradually decreased. Before treatment, IL-7 serum levels of patients were significantly higher than that in the control group (p < 0.01). After seven years of effective HAART, IL-7 levels had gradually decreased, but were still higher than in the control group (p < 0.01). The CD127 expression on CD3+ CD8+ T cells in effective HAART patients was higher than in untreated HIV+ patients (p < 0.05), but was lower than that in the control group (p < 0.05). CD127 expression on CD3+ CD4+ T cells was not significantly different among the control group, untreated HIV+ patients and effective HAART group. CONCLUSION: After seven years of effective HAART, the quantity and capacity of T cell subsets and IL-7 in HIV-1-infected patients had been partially restored, and the abnormal immune activation has significantly diminished.

Solitary intracerebral chondroma without meningeal attachment: a case report with review of the literature.

Intracranial chondromas are rare, benign cartilaginous tumours that account for < 0.3% of primary intracranial tumours. They usually originate from the basal synchondrosis and are extradural though, extremely rarely, they can be intracerebral. Here the case of a 45-year old female is presented with a solitary intracerebral chondroma located in the right frontal lobe with no meningeal attachment. The epidemiology, aetiology, clinical behaviour, radiological features, histological features and treatment of the case are discussed with a review of previous cases reported in the literature.

Interplay between telomere length and telomerase in human leukocyte differentiation and aging.

Blood leukocytes derive from bone marrow hematopoietic stem cells and differentiate into multiple types of mature cells that include granulocytes, monocytes, mast cells of myeloid lineage, and T and B lymphocytes of lymphoid lineage. Their distinctive paths of differentiation and unique roles in immune response provide a model for comparative analysis of biological parameters, such as telomere length and telomerase activity, in different types of leukocytes. Age has also been associated with the decline in immune functions and with the attrition of telomere length in leukocytes. This review will summarize recent progress in the study of telomere length and telomerase expression in leukocytes during differentiation and aging. In addition, I will attempt to shed new light on the roles of telomere and telomerase in leukocyte function and potential clinical interventions.

C. elegans CED-12 acts in the conserved crkII/DOCK180/Rac pathway to control cell migration and cell corpse engulfment.

We have identified and characterized a novel C. elegans gene, ced-12, that functions in the conserved GTPase signaling pathway mediated by CED-2/Crkll, CED-5/DOCK180, and CED-10/Rac to control cell migration and phagocytosis of apoptotic cells. We provide evidence that ced-12 likely acts upstream of ced-10 during cell migration and phagocytosis and that CED-12 physically interacts with CED-5 and forms a ternary complex with CED-2 in vitro. We propose that the formation and localization of a CED-2-CED-5-CED-12 ternary complex to the plasma membrane activates CED-10, leading to the cytoskeletal reorganization that occurs in the polarized extension of cell surfaces in engulfing cells and migrating cells. We suggest that CED-12 counterparts in higher organisms regulate cytoskeleton dynamics, as CED-12 does in C. elegans.

Identification of maze learning-associated genes in rat hippocampus by cDNA microarray.

Long-term memory formation requires de novo RNA and protein synthesis. To assess gene-expression changes associated with learning and memory processes, we used cDNA microarray to analyze hippocampal gene expression in male Fischer-344 rats following training in a multiunit T-maze. Here, we report the identification of 28 clones (18 known genes and 10 ESTs) for which expression increased after the maze learning. Some of the known genes appear to be involved in Ca2+ signaling, Ras activation, kinase cascades, and extracellular matrix (ECM) function, which may regulate neural transmission, synaptic plasticity, and neurogenesis. The gene-expression profile presented here provides the groundwork for future, more focused research to elucidate the contribution of these genes in learning and memory processes.

[The morphologic structure of vitreous hemorrhage and posterior vitreous detachment].

OBJECTIVE: To investigate the morphologic structures of vitreous hemorrhage that may be beneficial to its treatment. METHODS: The clinical data of 74 cases (79 eyes) with vitreous hemorrhage resulting from proliferative diabetic retinopathy, retinal vasculitis and retinal vein occlusion in December 1995 to January 2000 and having undergone vitrectomy were studied retrospectively, which focused on the morphologic characteristics of vitreous body by B-scan, surgical microscopy and surgical manipulation, to evaluate the relation of retina and vitreous body in order to discern the relationships among the retina, neovascular fibromembrane and the posterior hyaloid surface. RESULTS: Posterior vitreous detachment (PVD) in various degrees was observed in all the cases. The morphologic character was classified as the complete and partial vitreous detachment. The latter one can be subdivided into "V", "L" types and posterior vitreoschisis that was not rarely seen in the proliferative phase of ischemic retinopathy. CONCLUSIONS: To evaluate the characteristics of posterior vitreous detachment and vitreoschisis may provide valuable information for vitreoretinal surgery, elevate the rate of success and improve the prognosis of the surgery.

The role of telomerase expression and telomere length maintenance in human and mouse.

The molecular regulation of telomere length has been well elucidated by a series of elegant studies over the past decade. More recently, experimental evidence has accrued that addresses the challenging question of if and how telomere length regulation may contribute to normal human aging or to human disease. Recent studies in mice have provided a mammalian precedent indicating that telomerase deficiency can lead to in vivo dysfunction, most probably as a consequence of progressive telomere shortening. In humans, the evidence that telomere shortening might lead to in vivo dysfunction is far less direct, although the recent description of telomerase deficiency and telomere shortening associated with the DKC syndrome is suggestive of such a link. Methodologies exist and continue to be developed that are increasingly capable of manipulating telomerase activity and telomere length in human cells. It remains to be determined whether scientifically rigorous and (equally important) medically ethical approaches will emerge to directly assess the ability of telomere length modulation to correct functional disorders of human cellular function ex vivo or more challenging still, in vivo.

Lineage-specific telomere shortening and unaltered capacity for telomerase expression in human T and B lymphocytes with age.

Age effects on telomere length and telomerase expression in peripheral blood lymphocytes were analyzed from 121 normal individuals age newborn to 94 years and revealed several new findings. 1) Telomere shortening was observed in CD4+ and CD8+ T and B cells with age. However, the rate of telomere loss was significantly different in these populations, 35 +/- 8, 26 +/- 7, and 19 +/- 7 bp/year for CD4+ and CD8+ T and B cells, respectively. In addition, CD4+ T cells had the longest average telomeres at all ages, followed by B cells, with CD8+ T cell telomeres the shortest, suggesting that these lymphocyte populations may have different replicative histories in vivo. 2) Telomerase activity in freshly isolated T and B cells was indistinguishably low to undetectable at all ages but was markedly increased after Ag and costimulatory receptors mediated stimulation in vitro. Furthermore, age did not alter the magnitude of telomerase activity induced after stimulation of T or B lymphocytes through Ag and costimulatory receptors or in response to PMA plus ionomycin treatment. 3) The levels of telomerase activity induced by in vitro stimulation varied among individual donors but were highly correlated with the outcome of telomere length change in CD4+ T cells after Ag receptor-mediated activation. Together, these results indicate that rates of age-associated loss of telomere length in vivo in peripheral blood lymphocytes is specific to T and B cell subsets and that age does not significantly alter the capacity for telomerase induction in lymphocytes.

Constitutive and regulated expression of telomerase reverse transcriptase (hTERT) in human lymphocytes.

Human telomerase consists of two essential components, telomerase RNA template (hTER) and telomerase reverse transcriptase (hTERT), and functions to synthesize telomere repeats that serve to protect the integrity of chromosomes and to prolong the replicative life span of cells. Telomerase activity is expressed selectively in germ-line and malignant tumor cells but not in most normal human somatic cells. As a notable exception, telomerase is expressed in human lymphocytes during development, differentiation, and activation. Recent studies have suggested that regulation of telomerase is determined by transcription of hTERT but not hTER. The highly regulated expression of telomerase in lymphocytes provides an opportunity to analyze the contribution of transcriptional regulation of hTERT and hTER. We report here an analysis of hTERT expression by Northern and in situ hybridization. It was found that hTERT mRNA is expressed at detectable levels in all subsets of human lymphocytes isolated from thymus, tonsil, and peripheral blood, regardless of the status of telomerase activity. hTERT expression is regulated as a function of lineage development, differentiation, and activation. Strikingly, however, telomerase activity in these cells is not correlated strictly with the levels of hTERT and hTER transcripts. The absence of correlation between telomerase activity and hTERT mRNA could not be attributed to the presence of hTERT splice variants or to detectable inhibitors of telomerase activity. Thus, transcriptional regulation of hTERT is not sufficient to account for telomerase activity in human lymphocytes, indicating a likely role of posttranscriptional factors in the control of enzyme function.

Cutting edge: antigen-dependent regulation of telomerase activity in murine T cells.

Telomeres, structures on the ends of linear chromosomes, function to maintain chromosomal integrity. Telomere shortening occurs with cell division and provides a mechanism for limiting the replicative potential of normal human somatic cells. Telomerase, a ribonucleoprotein enzyme, synthesizes telomeric repeats on chromosomal termini, potentially extending the capacity for cell division. The present study demonstrates that resting T cells express little/no activity, and optimal Ag-specific induction of telomerase activity in vitro requires both TCR and CD28-B7 costimulatory signals. Regulation of telomerase in T cells during in vivo Ag-dependent activation was also assessed by adoptive transfer of TCR transgenic T cells and subsequent Ag challenge. Under these conditions, telomerase was induced in transgenic T cells coincident with a phase of extensive clonal expansion. These findings suggest that telomerase may represent an adoptive response that functions to preserve replicative potential in Ag-reactive lymphocytes.

[The clinical features of retinal detachment with congenital choroidal coloboma].

OBJECTIVE: To approach the clinical features of retinal detachment with congenital choroidal coloboma and seek better surgical methods to improve the effect of treatment. METHODS: Vitreoretinal surgery was performed on eight patients of retinal detachment with choroidal coloboma. During surgery by using an operating microscope with high magnification and endoillumination, we searched for retinal break(s) located in the colobomatous area, looked into the condition of posterior hyaloid area, status of coloboma, treatment was given accordingly, and finally silicone oil tamponade was performed. RESULTS: Retina was reattached anatomically after operation in all cases, and their visual acuities improved. CONCLUSIONS: The type of retinal detachment is caused by the retinal break inside the coloboma. The breaks located at the edge of the detachment within the coloboma, ectatic sclera at the colobomatous region, without posterior vitreous detachment are the features of the retinal detachment with congenital choroidal coloboma. Complete vitrectomy with method to create chorioretinal adhesion around the coloboma and silicone oil tamponade provide an effective treatment for this complicated type of retinal detachment.

[A preliminary investigation on causes of failure of vitreoretinal surgery for complicated rhegmatogenic retinal detachments].

OBJECTIVE: To evaluate the failure causes of vitreoretinal surgery for complicated rhegmatogenic retinal detachment. METHODS: Vitreoretinal (VR) surgery was performed on 477 cases (479 eyes) with complicated rhegmatogenic retinal detachment, the operative procedures including vitrectomy, peeling of pre-retinal membrane, fluid-air exchange, inert gas (SF(6) or C(3)F(8)) or silicone tamponade. RESULTS: On discharge, the operation was effective in 349 eyes (72.87%) and failed in 130 (27.13%) eyes. CONCLUSION: The multiple stepwise multivariate regression shows that the factors significantly influencing VR surgery are silicone tamponade, giant retinal tears, SF(6) tamponade, iatrogenic tear, anterior proliferative vitreoretinopathy (PVR), subretinal proliferation, PVR, C(3)F(8) tamponade, operative frequency and choroidal detachments.

Three-dimensional surface reconstruction using optical flow for medical imaging.

The recovery of a three-dimensional (3-D) model from a sequence of two-dimensional (2-D) images is very useful in medical image analysis. Image sequences obtained from the relative motion between the object and the camera or the scanner contain more 3-D information than a single image. Methods to visualize the computed tomograms can be divided into two approaches: the surface rendering approach and the volume rendering approach. In this paper, a new surface rendering method using optical flow is proposed. Optical flow is the apparent motion in the image plane produced by the projection of the real 3-D motion onto 2-D image. The 3-D motion of an object can be recovered from the optical-flow field using additional constraints. By extracting the surface information from 3-D motion, it is possible to get an accurate 3-D model of the object. Both synthetic and real image sequences have been used to illustrate the feasibility of the proposed method. The experimental results suggest that the proposed method is suitable for the reconstruction of 3-D models from ultrasound medical images as well as other computed tomograms.

Telomere lengthening and telomerase activation during human B cell differentiation.

The function of the immune system is highly dependent on cellular differentiation and clonal expansion of antigen-specific lymphocytes. However, little is known about mechanisms that may have evolved to protect replicative potential in actively dividing lymphocytes during immune differentiation and response. Here we report an analysis of telomere length and telomerase expression, factors implicated in the regulation of cellular replicative lifespan, in human B cell subsets. In contrast to previous observations, in which telomere shortening and concomitant loss of replicative potential occur in the process of somatic cell differentiation and cell division, it was found that germinal center (GC) B cells, a compartment characterized by extensive clonal expansion and selection, had significantly longer telomeric restriction fragments than those of precursor naive B cells. Furthermore, it was found that telomerase, a telomere-synthesizing enzyme, is expressed at high levels in GC B cells (at least 128-fold higher than those of naive and memory B cells), correlating with the long telomeres in this subset of B cells. Finally, both naive and memory B cells were capable of up-regulating telomerase activity in vitro in response to activation signals through the B cell antigen receptor in the presence of CD40 engagement and/or interleukin 4. These observations suggest that a novel process of telomere lengthening, possibly mediated by telomerase, functions in actively dividing GC B lymphocytes and may play a critical role in humoral immune response by maintaining the replicative potential of GC and descendant memory B cells.

Regulation of telomerase RNA template expression in human T lymphocyte development and activation.

Telomeres are unique DNA-protein complexes at the terminals of chromosomes that appear to play a critical role in protecting chromosomal integrity and in maintaining cellular replicative potential. Telomerase is a ribonuclear protein that is capable of elongating telomeres by the addition of telomeric hexanucleotide repeats and therefore contributing to the capacity for cell replication. Telomerase activity is expressed in human germline cells and malignant cells, and it has recently been demonstrated that telomerase activity is highly regulated in normal lymphocytes at specific stages of development and activation. However, these studies have not elucidated whether telomerase activity is regulated at the level of specific gene expression or whether the regulation of telomerase RNA template (hTR) and/or protein components contributes to the regulation of telomerase activity in normal somatic cells. To characterize at a molecular level the regulation of telomerase expression in human T lymphocytes, we analyzed the expression of hTR during lineage development and after in vitro activation. It was found that hTR is expressed in subsets of thymocytes with strong telomerase activity at levels that are consistently higher (1.5 times; p < 0.01) than those found in peripheral blood resting T cells. In addition, hTR is up-regulated two- to fivefold in peripheral blood naive and memory CD4+ T cells after in vitro activation with anti-CD3 plus anti-CD28. These results establish that hTR expression is regulated in normal human T cells during lineage development and after activation, and indicate that regulation of hTR expression may contribute to the regulation of telomerase activity in normal lymphoid cells.