Long-term antiretroviral treatment initiated at primary HIV-1 infection affects the size, composition, and decay kinetics of the reservoir of HIV-1-infected CD4 T cells.

Initiation of antiretroviral therapy during the earliest stages of HIV-1 infection may limit the seeding of a long-lasting viral reservoir, but long-term effects of early antiretroviral treatment initiation remain unknown. Here, we analyzed immunological and virological characteristics of nine patients who started antiretroviral therapy at primary HIV-1 infection and remained on suppressive treatment for >10 years; patients with similar treatment duration but initiation of suppressive therapy during chronic HIV-1 infection served as controls. We observed that independently of the timing of treatment initiation, HIV-1 DNA in CD4 T cells decayed primarily during the initial 3 to 4 years of treatment. However, in patients who started antiretroviral therapy in early infection, this decay occurred faster and was more pronounced, leading to substantially lower levels of cell-associated HIV-1 DNA after long-term treatment. Despite this smaller size, the viral CD4 T cell reservoir in persons with early treatment initiation consisted more dominantly of the long-lasting central-memory and T memory stem cells. HIV-1-specific T cell responses remained continuously detectable during antiretroviral therapy, independently of the timing of treatment initiation. Together, these data suggest that early HIV-1 treatment initiation, even when continued for >10 years, is unlikely to lead to viral eradication, but the presence of low viral reservoirs and durable HIV-1 T cell responses may make such patients good candidates for future interventional studies aiming at HIV-1 eradication and cure. IMPORTANCE: Antiretroviral therapy can effectively suppress HIV-1 replication to undetectable levels; however, HIV-1 can persist despite treatment, and viral replication rapidly rebounds when treatment is discontinued. This is mainly due to the presence of latently infected CD4 T cells, which are not susceptible to antiretroviral drugs. Starting treatment in the earliest stages of HIV-1 infection can limit the number of these latently infected cells, raising the possibility that these viral reservoirs are naturally eliminated if suppressive antiretroviral treatment is continued for extremely long periods of time. Here, we analyzed nine patients who started on antiretroviral therapy within the earliest weeks of the disease and continued treatment for more than 10 years. Our data show that early treatment accelerated the decay of infected CD4 T cells and led to very low residual levels of detectable HIV-1 after long-term therapy, levels that were otherwise detectable in patients who are able to maintain a spontaneous, drug-free control of HIV-1 replication. Thus, long-term antiretroviral treatment started during early infection cannot eliminate HIV-1, but the reduced reservoirs of HIV-1 infected cells in such patients may increase their chances to respond to clinical interventions aiming at inducing a drug-free remission of HIV-1 infection.

The oxidative potential of differently charged silver and gold nanoparticles on three human lung epithelial cell types

Background: Nanoparticle (NPs) functionalization has been shown to affect their cellular toxicity. To study this, differently functionalized silver (Ag) and gold (Au) NPs were synthesised, characterised and tested using lung epithelial cell systems. Mehtods: Monodispersed Ag and Au NPs with a size range of 7 to 10 nm were coated with either sodium citrate or chitosan resulting in surface charges from ¿50 mV to +70 mV. NP-induced cytotoxicity and oxidative stress were determined using A549 cells, BEAS-2B cells and primary lung epithelial cells (NHBE cells). TEER measurements and immunofluorescence staining of tight junctions were performed to test the growth characteristics of the cells. Cytotoxicity was measured by means of the CellTiter-Blue ® and the lactate dehydrogenase assay and cellular and cell-free reactive oxygen species (ROS) production was measured using the DCFH-DA assay. Results: Different growth characteristics were shown in the three cell types used. A549 cells grew into a confluent mono-layer, BEAS-2B cells grew into a multilayer and NHBE cells did not form a confluent layer. A549 cells were least susceptible towards NPs, irrespective of the NP functionalization. Cytotoxicity in BEAS-2B cells increased when exposed to high positive charged (+65-75 mV) Au NPs. The greatest cytotoxicity was observed in NHBE cells, where both Ag and Au NPs with a charge above +40 mV induced cytotoxicity. ROS production was most prominent in A549 cells where Au NPs (+65-75 mV) induced the highest amount of ROS. In addition, cell-free ROS measurements showed a significant increase in ROS production with an increase in chitosan coating. Conclusions: Chitosan functionalization of NPs, with resultant high surface charges plays an important role in NP-toxicity. Au NPs, which have been shown to be inert and often non-cytotoxic, can become toxic upon coating with certain charged molecules. Notably, these effects are dependent on the core material of the particle, the cell type used for testing and the growth characteristics of these cell culture model systems.

Primary γδ T cell lymphoma of the lung: report of a case with features suggesting derivation from intraepithelial γδ T lymphocytes [Primary gammadelta T cell lymphoma of the lung: report of a case with features suggesting derivation from intraepithelial gammadelta T lymphocytes].

T cell lymphoma of γδ T cell origin is a rare disease that mainly involves extranodal sites and shows aggressive clinical behavior. Here, we report a case of primary γδ T cell lymphoma of the lungs with epitheliotropism in the respiratory epithelium, a feature somewhat reminiscent of what is observed in enteropathy-associated T cell lymphoma. A 63-year-old man presented with chest pain and dyspnea on exertion, weight loss, and general weakness. On a positron emission tomography (PET) scan, multiple hypermetabolic lesions were found in both lungs. Microscopic examination of the wedge lung biopsy revealed nodular infiltration of monomorphic, medium- to large-sized atypical lymphocytes with round nuclei, coarse chromatin, and a variable amount of clear to eosinophilic cytoplasm. Of note, intraepithelial lymphocytosis by atypical lymphoid cells was observed in the respiratory epithelium within and around the nodule. Immunohistochemically, the tumor cells were CD3+, TCRβF1-, TCRγ+, CD5-, CD7+, CD20-, CD79a-, CD30-, CD4-, CD8-, CD10-, BCL6-, CD21-, CD56+, CD57-, and CD138-, and expressed cytotoxic molecules. Epstein-Barr virus (EBV) was not detected by an in situ hybridization assay for EBV-encoded RNA. Interestingly, CD103 was expressed by a subset of tumor cells, especially those infiltrating the epithelium. T cell clonality was detected by multiplex PCR analysis of TRG and TRD gene rearrangements. After 2 months of systemic chemotherapy, PET scan showed regression of the size and metabolic activity of the lesions. This case represents a unique γδ T cell lymphoma of the lungs showing epitheliotropism by CD103+ γδ T cells that is suggestive of tissue-resident γδ T cells as the cell of origin.

The DNA damage response to adeno-associated virus : centrosome overduplication and induction of cell death independently of the spindle checkpoint

Summary: Adeno-associated virus type 2 (AAV2) is a small virus containing single-stranded DNA of approximately 4.7kb in size. Both ends of the viral genome are flanked with inverted terminal repeat sequences (ITRs), which serve as primers for viral replication. Previous work in our laboratory has shown that AAV2 DNA with ultraviolet radiation-generated crosslinks (UV-AAV2) provokes a DNA damage response in the host cell by mimicking a stalled replication fork. Infection of cells with UV-AAV2 leads to a p53-and Chk1-mediated cell cycle arrest at the G2/M border of the cell cycle. However, tumour cells lacking the tumour suppressor protein p53 cannot sustain this arrest and enter a prolonged impaired mitosis, the outcome of which is cell death. The aim of my thesis was to investigate how UV-inactivated AAV2 kilts p53-deficient cancer cells. I found that the UV-AAV2-induced DNA damage signalling induces centriole overduplication in infected cells. The virus is able to uncouple the centriole duplication cycle from the cell cycle, leading to amplified centrosome numbers. Chk1 colocalises with centrosomes in the infected cells and the centrosome overduplication is dependent on the presence of Chk1, as well as on the activities of ATR and Cdk kinases and on the G2 arrest. The UV-AAV2-induced DNA damage signalling inhibits the degradation of cyclin B 1 and securin by the anaphase promoting complex, suggesting that the spindle checkpoint is activated in these mitotic cells. Interference with the spindle checkpoint components Mad2 and BubR1 revealed that the UV-AAV2-provoked mitotic catastrophe occurs independently of spindle checkpoint function, This work shows that, in the p53 deficient cells, UV-AAV2 triggers mitotic catastrophe associated with a dramatic Chk1-dependent overduplication of centrioles and the consequent formation of multiple spindle poles in mitosis. Résumé Le virus associé à l'adénovirus type 2 (AAV2) est un petit virus contenant un simple brin d'ADN d'environ 4.7kb. Des expériences antérieures dans notre laboratoire ont montré que les liens intramoléculaires sur l'ADN de AAV2 provoqués paz l'irradiation aux ultraviolets (UV) ressemblent à une fourche de réplication bloquée, ce qui provoque une réponse aux dommages à l'ADN dans la cellule hôte. L'infection des cellules avec UV-AAV2 résulte en un arrêt du cycle cellulaire à la transition G2/M entraîné par les protéines ATR et Chk1. Cependant, les cellules tumorales auxquelles il manque le suppresseur de tumeur p53 ne peuvent pas tenir cet arrêt et entrent dans une mitose anormale et prolongée qui se terminera par la mort cellulaire. Le but de ma thèse était d'étudier comment l'AAV2 inactivé par l'irradiation UV tue les cellules cancéreuses n'ayant pas p53. Je montre ici que le signal de dommages à l'ADN induit par UV-AAV2 génère une surduplication des centrioles dans les cellules infectées. Le virus est capable de dissocier le cycle de duplication du centriole du cycle cellulaire ce qui crée un nombre amplifié de centrosomes. Chk1 est co-localisé avec le centrosome dans les cellules infectées et la swduplication du centrosome est dépendante de la présence de Chk1, de l'activité des kinases ATR et Cdk et de l'arrêt en G2 de la cellule. Le signal d'ADN endommagé induit par UV-AAV2 réprime la dégradation des protéines cycline B1 et securine par le complexe promoteur de l'anaphase (APC), ce qui suggère que le point de contrôle du fuseau mitotique est activé dans ces cellules en mitose. L'étude d'interférence avec des éléments du point de contrôle du fuseau mitotique, Mad2 et BubR1, a révélé que la catastrophe mitotique provoquée paz UV-AAV2 survient indépendamment du point de contrôle du fuseau mitotique. Ce travail montre que dans les cellules déficientes en p53, UV-AAV2 induit une catastrophe mitotique associée à une surduplication des centrioles dépendant de Chk1 et ayant pour conséquence dramatique la formation de multiples fuseaux mitotiques dans la cellule en mitose.

Fibrosarcoma-like lipomatous neoplasm: a reappraisal of so-called spindle cell liposarcoma defining a unique lipomatous tumor unrelated to other liposarcomas

The term "spindle cell liposarcoma" has been applied to liposarcomas (LPSs) composed predominantly or exclusively of spindled cells. These tumors have been considered variants of well-differentiated LPS (WDL), myxoid LPS, and spindle cell lipoma, suggesting that this is a heterogenous group of lesions. Using strict morphologic criteria and molecular and immunohistochemical analyses, we have identified a homogenous group of spindle cell lipomatous tumors, histologically and genetically distinct from other forms of LPS, which we have called "fibrosarcoma-like lipomatous neoplasm." Cases classified as "spindle cell LPS" or "low-grade LPS with spindle cell features" were reviewed. Final selection criteria included: (1) an exclusive low-grade spindle cell component resembling fibrosarcoma; (2) a mixture of bland fibroblastic cells resembling the preadipocyte and early-adipocyte stage of embryonic fat; and (3) molecular-genetic analysis that excluded other forms of lipomatous tumors. Of the initial 25 cases identified, comparative genomic hybridization (CGH) was uninformative in 2 cases; 5 were reclassified as WDL on the basis of molecular data (MDM2 amplification) and 6 as spindle cell lipoma (CGH profiles with a few gains and losses including a constant loss of chromosome 13 and frequent losses of chromosomes 16 and 6). The 12 remaining cases showed flat CGH profiles; of these cases, 11 were negative for DDIT3 gene rearrangements, and 1 result was uninterpretable. Patients ranged in age from 15 to 82 years (mean 50 y); male patients were affected slightly more often (7:5). Tumors arose in the deep (6) and superficial (3) soft tissue of the groin (4), buttock (3), thigh (2), flank (1), shoulder (1), and paratesticular tissue (1) and ranged in size from 2 to 20 cm (mean 7.5 cm). Clinical follow-up in 11 patients (9 mo to 20 y; mean 68 mo) showed no recurrences or metastases. As defined above, "fibrosarcoma-like lipomatous neoplasm" is a unique lipomatous tumor that should be distinguished from WDL/(low-grade) dedifferentiated LPS and myxoid LPS on combined histologic/molecular features because of its better prognosis.

Comparison of the prognostic value of pretreatment measurements of systemic inflammatory response in patients undergoing curative resection of clear cell renal cell carcinoma.

PURPOSE: Pretreatment measurements of systemic inflammatory response, including the Glasgow prognostic score (GPS), the neutrophil-to-lymphocyte ratio (NLR), the monocyte-to-lymphocyte ratio (MLR), the platelet-to-lymphocyte ratio (PLR) and the prognostic nutritional index (PNI) have been recognized as prognostic factors in clear cell renal cell carcinoma (CCRCC), but there is at present no study that compared these markers. METHODS: We evaluated the pretreatment GPS, NLR, MLR, PLR and PNI in 430 patients, who underwent surgery for clinically localized CCRCC (pT1-3N0M0). Associations with disease-free survival were assessed with Cox models. Discrimination was measured with the C-index, and a decision curve analysis was used to evaluate the clinical net benefit. RESULTS: On multivariable analyses, all measures of systemic inflammatory response were significant prognostic factors. The increase in discrimination compared with the stage, size, grade and necrosis (SSIGN) score alone was 5.8 % for the GPS, 1.1-1.4 % for the NLR, 2.9-3.4 % for the MLR, 2.0-3.3 % for the PLR and 1.4-3.0 % for the PNI. On the simultaneous multivariable analysis of all candidate measures, the final multivariable model contained the SSIGN score (HR 1.40, P < 0.001), the GPS (HR 2.32, P < 0.001) and the MLR (HR 5.78, P = 0.003) as significant variables. Adding both the GPS and the MLR increased the discrimination of the SSIGN score by 6.2 % and improved the clinical net benefit. CONCLUSIONS: In patients with clinically localized CCRCC, the GPS and the MLR appear to be the most relevant prognostic measures of systemic inflammatory response. They may be used as an adjunct for patient counseling, tailoring management and clinical trial design.

Optimized exosome isolation protocol for cell culture supernatant and human plasma.

Extracellular vesicles represent a rich source of novel biomarkers in the diagnosis and prognosis of disease. However, there is currently limited information elucidating the most efficient methods for obtaining high yields of pure exosomes, a subset of extracellular vesicles, from cell culture supernatant and complex biological fluids such as plasma. To this end, we comprehensively characterize a variety of exosome isolation protocols for their efficiency, yield and purity of isolated exosomes. Repeated ultracentrifugation steps can reduce the quality of exosome preparations leading to lower exosome yield. We show that concentration of cell culture conditioned media using ultrafiltration devices results in increased vesicle isolation when compared to traditional ultracentrifugation protocols. However, our data on using conditioned media isolated from the Non-Small-Cell Lung Cancer (NSCLC) SK-MES-1 cell line demonstrates that the choice of concentrating device can greatly impact the yield of isolated exosomes. We find that centrifuge-based concentrating methods are more appropriate than pressure-driven concentrating devices and allow the rapid isolation of exosomes from both NSCLC cell culture conditioned media and complex biological fluids. In fact to date, no protocol detailing exosome isolation utilizing current commercial methods from both cells and patient samples has been described. Utilizing tunable resistive pulse sensing and protein analysis, we provide a comparative analysis of 4 exosome isolation techniques, indicating their efficacy and preparation purity. Our results demonstrate that current precipitation protocols for the isolation of exosomes from cell culture conditioned media and plasma provide the least pure preparations of exosomes, whereas size exclusion isolation is comparable to density gradient purification of exosomes. We have identified current shortcomings in common extracellular vesicle isolation methods and provide a potential standardized method that is effective, reproducible and can be utilized for various starting materials. We believe this method will have extensive application in the growing field of extracellular vesicle research.

JNK controls the onset of mitosis of planarian stem cells and triggers apoptotic cell death required for regeneration and remodeling

Regeneration of lost tissues depends on the precise interpretation of molecular signals that control and coordinate the onset of proliferation, cellular differentiation and cell death. However, the nature of those molecular signals and the mechanisms that integrate the cellular responses remain largely unknown. The planarian flatworm is a unique model in which regeneration and tissue renewal can be comprehensively studied in vivo. The presence of a population of adult pluripotent stem cells combined with the ability to decode signaling after wounding enable planarians to regenerate a complete, correctly proportioned animal within a few days after any kind of amputation, and to adapt their size to nutritional changes without compromising functionality. Here, we demonstrate that the stress-activated c-jun-NH2-kinase (JNK) links wound-induced apoptosis to the stem cell response during planarian regeneration. We show that JNK modulates the expression of wound-related genes, triggers apoptosis and attenuates the onset of mitosis in stem cells specifically after tissue loss. Furthermore, in pre-existing body regions, JNK activity is required to establish a positive balance between cell death and stem cell proliferation to enable tissue renewal, remodeling and the maintenance of proportionality. During homeostatic degrowth, JNK RNAi blocks apoptosis, resulting in impaired organ remodeling and rescaling. Our findings indicate that JNK-dependent apoptotic cell death is crucial to coordinate tissue renewal and remodeling required to regenerate and to maintain a correctly proportioned animal. Hence, JNK might act as a hub, translating wound signals into apoptotic cell death, controlled stem cell proliferation and differentiation, all of which are required to coordinate regeneration and tissue renewal.

Gleevec, an Abl family inhibitor, produces a profound change in cell shape and migration (in press)

The issue of how contractility and adhesion are related to cell shape and migration pattern remains largely unresolved. In this paper we report that Gleevec (Imatinib), an Abl family kinase inhibitor, produces a profound change in the shape and migration of rat bladder tumor cells (NBTII) plated on collagen-coated substrates. Cells treated with Gleevec adopt a highly spread D-shape and migrate more rapidly with greater persistence. Accompanying this more spread state is an increase in integrin-mediated adhesion coupled with increases in the size and number of discrete adhesions. In addition, both total internal reflection fluorescence microscopy (TIRFM) and interference reflection microscopy (IRM) revealed a band of small punctate adhesions with rapid turnover near the cell leading margin. These changes led to an increase in global cell-substrate adhesion strength, as assessed by laminar flow experiments. Gleevec-treated cells have greater RhoA activity which, via myosin activation, led to an increase in the magnitude of total traction force applied to the substrate. These chemical and physical alterations upon Gleevec treatment produce the dramatic change in morphology and migration that is observed.

Nuclear Matrix in Apoptotic Cell Death and Cell Proliferation. The role of Nuclear Mitotic Apparatus (NuMA) Protein

The nucleus is a membrane enclosed organelle containing most of the genetic information of the cell in the form of chromatin. The nucleus, which can be divided into many sub-organelles such as the nucleoli, the Cajal bodies and the nuclear lamina, is the site for several essential cellular functions such as the DNA replication and its regulation and most of the RNA synthesis and processing. The nucleus is often affected in disease: the size and the shape of the nucleus, the chromatin distribution and the size of the nucleoli have remained the basis for the grading of several cancers. The maintenance of the vertebrate body shape depends on the skeleton. Similarly, in a smaller context, the shape of the cell and the nucleus are mainly regulated by the cytoskeletal and nucleoskeletal elements. The nuclear matrix, which by definition is a detergent, DNase and salt resistant proteinaceous nuclear structure, has been suggested to form the nucleoskeleton responsible for the nuclear integrity. Nuclear mitotic apparatus protein, NuMA, a component of the nuclear matrix, is better known for its mitotic spindle organizing function. NuMA is one of the nuclear matrix proteins suggested to participate in the maintenance of the nuclear integrity during interphase but its interphase function has not been solved to date. This thesis study concentrated on the role of NuMA and the nuclear matrix as structural and functional components of the interphase nucleus. The first two studies clarified the essential role of caspase-3 in the disintegration of the nuclear structures during apoptosis. The second study also showed NuMA and chromatin to co-elute from cells in significant amounts and the apoptotic cleavage of NuMA was clarified to have an important role in the dissociation of NuMA from the chromatin. The third study concentrated on the interphase function of NuMA showing NuMA depletion to result in cell cycle arrest and the cytoplasmic relocalization of NuMA interaction partner GAS41. We suggest that the relocalization of the transcription factor GAS41 may mediate the cell cycle arrest. Thus, this study has given new aspects in the interactions of NuMA, chromatin and the nuclear matrix.

System-level characterization of Th2 cell development and immune cell responses to ZnO and TiO2 nanoparticles

Asthma and allergy are common diseases and their prevalence is increasing. One of the hypotheses that explains this trend is exposure to inhalable chemicals such as traffi c-related air pollution. Epidemiological research supports this theory, as a correlation between environmental chemicals and allergic respiratory diseases has been found. In addition to ambient airborne particles, one may be exposed to engineered nanosized materials that are actively produced due to their favorable physico-chemical properties compared to their bulk size counterparts. On the cellular level, improper activity of T helper (Th) cells has been connected to allergic reactions. Th cells can differentiate into functionally different effector subsets, which are identifi ed according to their characteristic cytokine profi les resulting in specifi c ability to communicate with other cells. Th2 cells activate humoral immunity and stimulate eradication of extracellular pathogens. However, persistent predominance of Th2 cells is involved in a development of number of allergic diseases. The cytokine environment at the time of antigen recognition is the major factor determining the polarization of a naïve Th cell. Th2 cell differentiation is initiated by IL4, which signals via transcription factor STAT6. Although the importance of this pathway has been evaluated in the mouse studies, the signaling components involved have been largely unknown. The aim of this thesis was to identify molecules, which are under the control of IL4 and STAT6 in Th cells. This was done by using system-level analysis of STAT6 target genes at genome, mRNA and protein level resulting in identifi cation of various genes previously not connected to Th2 cell phenotype acquisition. In the study, STAT6-mediated primary and secondary target genes were dissection from each other and a detailed transcriptional kinetics of Th2 cell polarization of naïve human CD4+ T cells was collected. Integration of these data revealed the hierarchy of molecular events that mediates the differentiation towards Th2 cell phenotype. In addition, the results highlighted the importance of exploiting proteomics tools to complement the studies on STAT6 target genes identifi ed through transcriptional profi ling. In the last subproject, the effects of the exposure with ZnO and TiO2 nanoparticles was analyzed in Jurkat T cell line and in primary human monocyte-derived macrophages and dendritic cells to evaluate their toxicity and potential to cause infl ammation. Identifi cation of ZnO-derived gene expression showed that the same nanoparticles may elicit markedly distinctive responses in different cell types, thus underscoring the need for unbiased profi ling of target genes and pathways affected. The results gave additional proof that the cellular response to nanosized ZnO is due to leached Zn2+ ions. The approach used in ZnO and TiO2 nanoparticle study demonstrated the value of assessing nanoparticle responses through a toxicogenomics approach. The increased knowledge of Th2 cell signaling will hopefully reveal new therapeutic nodes and eventually improve our possibilities to prevent and tackle allergic infl ammatory diseases.

Is there correlation between the turbulent eddies size and mechanical hemolysis ?

Hemolytic profile of an artificial device chronically implanted in the cardiovascular system may represent the difference between the success and failure in its long-term performance. Last decades have witnessed efforts on the development of methods capable of predicting red blood cell damage in artificial organs. However, all of them have had limited success to predict hemolysis. The primary cause of this problem is that such models do not take into consideration structures of turbulent flow. The present paper demonstrates that microscopic measurable occurrences of the turbulent flow may be linked to red blood cell trauma. This study suggests that if the smallest turbulent eddies dimension is under 10 m m hemolysis is not dependent on the exposure time and the red blood cells damage depends only on the dissipation of the turbulent energy in the erythrocyte membrane. The analysis reported here opens the possibility of mapping the flow field in artificial assist devices based on the smallest eddy length scales. This is a promising new trend and should be considered in the designing requirements of the next generations of artificial organs.

Horizontal Slug Flow in a Large-Size Pipeline: Experimentation and Modeling

The knowledge of the slug flow characteristics is very important when designing pipelines and process equipment. When the intermittences typical in slug flow occurs, the fluctuations of the flow variables bring additional concern to the designer. Focusing on this subject the present work discloses the experimental data on slug flow characteristics occurring in a large-size, large-scale facility. The results were compared with data provided by mechanistic slug flow models in order to verify their reliability when modelling actual flow conditions. Experiments were done with natural gas and oil or water as the liquid phase. To compute the frequency and velocity of the slug cell and to calculate the length of the elongated bubble and liquid slug one used two pressure transducers measuring the pressure drop across the pipe diameter at different axial locations. A third pressure transducer measured the pressure drop between two axial location 200 m apart. The experimental data were compared with results of Camargo's1 algorithm (1991, 1993), which uses the basics of Dukler & Hubbard's (1975) slug flow model, and those calculated by the transient two-phase flow simulator OLGA.

The molecular basis of selective permeability of connexins is complex and includes both size and charge

Although gap junction channels are still widely viewed as large, non-specific pores connecting cells, the diversity in the connexin family has led more attention to be focused on their permeability characteristics. We summarize here the current status of these investigations, both published and on-going, that reveal both charge and size selectivity between gap junction channels composed of different connexins. In particular, this review will focus on quantitative approaches that monitor the expression level of the connexins, so that it is clear that differences that are seen can be attributed to channel properties. The degree of selectivity that is observed is modest compared to other channels, but is likely to be significant for biological molecules that are labile within the cell. Of particular relevance to the in vivo function of gap junctions, recent studies are summarized that demonstrate that the connexin phenotype can control the nature of the endogenous traffic between cells, with consequent effects on biological effects of gap junctions such as tumor suppression.

Synthesis and secretion of transferrin by a bovine trabecular meshwork cell line

The trabecular meshwork (TM) is the main outflow pathway in the mammalian eye. Oxidative damage to TM cells has been suggested to be an important cause of impairment of TM functions, leading to deficient drainage of aqueous humor, with deleterious consequences to the eye. Transferrin, a metalloprotein involved in iron transport, has been characterized as an intrinsic eye protein. Since transferrin is implicated in the control of oxidative stress, the objective of the present study was to determine if a bovine TM cell line (CTOB) synthesizes and secretes transferrin. The CTOB cell line was cultured in the presence of 35S-methionine and the incubation medium was submitted to immunoprecipitation. Total RNAs from CTOB and isolated bovine TM (freshly isolated, incubated or not) were subjected to the reverse transcription-polymerase chain reaction and the amplification products were sequenced. Also, both CTOB and histological TM preparations were processed for transferrin immunolocalization. A labeled peptide of about 80 kDa, the expected size for transferrin, was immunopurified from CTOB samples obtained from the incubation assays. The reverse transcription-polymerase chain reaction and sequencing experiments detected the presence of transferrin mRNA in CTOB and isolated bovine TM. Reactivity to antibodies against transferrin was observed both in CTOB and TM. The results obtained in all of these experiments indicated that the TM is capable of synthesizing and secreting transferrin. The possible implications for the physiology of the eye are discussed.