Fred Hutchinson Cancer Research Center

About: Fred Hutchinson Cancer Research Center is a nonprofit organization based out in Cape Town, South Africa. It is known for research contribution in the topics: Population & Transplantation. The organization has 12322 authors who have published 30954 publications receiving 2288772 citations. The organization is also known as: Fred Hutch & The Hutch.

Overexpression of a kinase-inactive ATR protein causes sensitivity to DNA-damaging agents and defects in cell cycle checkpoints.

Abstract: ATR, a phosphatidylinositol kinase-related protein homologous to ataxia telangiectasia mutated (ATM), is important for the survival of human cells following many forms of DNA damage. Expression of a kinase-inactive allele of ATR (ATRkd) in human fibroblasts causes increased sensitivity to ionizing radiation (IR), cis-platinum and methyl methanesulfonate, but only slight UV radiation sensitivity. ATRkd overexpression abrogates the G2/M arrest after exposure to IR, and overexpression of wild-type ATR complements the radioresistant DNA synthesis phenotype of cells lacking ATM, suggesting a potential functional overlap between these proteins. ATRkd overexpression also causes increased sensitivity to hydroxyurea that is associated with microtubule-mediated nuclear abnormalities. These observations are consistent with uncoupling of certain mitotic events from the completion of S-phase. Thus, ATR is an important component of multiple DNA damage response pathways and may be involved in the DNA replication (S/M) checkpoint.

Lower Cancer Incidence in Amsterdam-I Criteria Families Without Mismatch Repair Deficiency: Familial Colorectal Cancer Type X

Abstract: ContextApproximately 60% of families that meet the Amsterdam-I criteria (AC-I) for hereditary nonpolyposis colorectal cancer (HNPCC) have a hereditary abnormality in a DNA mismatch repair (MMR) gene. Cancer incidence in AC-I families with MMR gene mutations is reported to be very high, but cancer incidence for individuals in AC-I families with no evidence of an MMR defect is unknown.ObjectiveTo determine if cancer risks in AC-I families with no apparent deficiency in DNA MMR are different from cancer risks in AC-I families with DNA MMR abnormalities.Design, Setting, and ParticipantsIdentification (1997-2001) of 161 AC-I pedigrees from multiple population- and clinic-based sources in North America and Germany, with families grouped into those with (group A) or without (group B) MMR deficiency by tumor testing. A total of 3422 relatives were included in the analyses.Main Outcome MeasuresCancer incidence in groups A and B (excluding the 3 affected members used to define each pedigree as AC-I) and computed age- and sex-adjusted standardized incidence ratios (SIRs) using Surveillance, Epidemiology, and End Results data.ResultsGroup A families from both population- and clinic-based series showed increased incidence of the HNPCC-related cancers. Group B families showed increased incidence only for colorectal cancer (SIR, 2.3; 95% confidence interval, 1.7-3.0) and to a lesser extent than group A (SIR, 6.1; 95% confidence interval, 5.2-7.2) (P<.001).ConclusionsFamilies who fulfill AC-I criteria but who have no evidence of a DNA MMR defect do not share the same cancer incidence as families with HNPCC-Lynch syndrome (ie, hereditary MMR deficiency). Relatives in such families have a lower incidence of colorectal cancer than those in families with HNPCC-Lynch syndrome, and incidence may not be increased for other cancers. These families should not be described or counseled as having HNPCC-Lynch syndrome. To facilitate distinguishing these entities, the designation of “familial colorectal cancer type X” is suggested to describe this type of familial aggregation of colorectal cancer.

Immune clearance of highly pathogenic SIV infection

Abstract: Established infections with the human and simian immunodeficiency viruses (HIV and SIV, respectively) are thought to be permanent with even the most effective immune responses and antiretroviral therapies only able to control, but not clear, these infections 1–4 .W hether the residual virus that maintains these infections is vulnerable to clearance is a question of central importance to the future management of millions of HIV-infected individuals. We recently reported that approximately 50% of rhesus macaques (RM; Macaca mulatta) vaccinated with SIV protein-expressing rhesus cytomegalovirus (RhCMV/ SIV) vectors manifest durable, aviraemic control of infection with the highly pathogenic strain SIVmac239 (ref. 5). Here we show that regardless of the route of challenge, RhCMV/SIV vector-elicited immune responses control SIVmac239 after demonstrable lymphatic and haematogenous viral dissemination, and that replicationcompetent SIV persists in several sites for weeks to months. Over time, however, protected RM lost signs of SIV infection, showing a consistent lack of measurable plasma- or tissue-associated virus using ultrasensitive assays, and a loss of T-cell reactivity to SIV determinants not in the vaccine. Extensive ultrasensitive quantitative PCR and quantitative PCR with reverse transcription analyses of tissues from RhCMV/SIV vector-protected RM necropsied 69–172 weeks after challenge did not detect SIV RNA or DNA sequences above background levels, and replication-competent SIV was not detected in these RM by extensive co-culture analysis of tissues or by adoptive transfer of 60 million haematolymphoid cells to naive RM. These data provide compelling evidence for progressive clearance of a pathogenic lentiviral infection, and suggest that some lentiviral reservoirs may be susceptible to the continuous effector memory T-cell-mediated immune surveillance elicited and maintained by cytomegalovirus vectors. Clinical and experimental observations have suggested that HIV and SIV infections might be vulnerable to immune control or pharmacological clearance in the first few hours to days of infection, before both the viral amplification needed for efficient mutational escape and the establishment of the highly resilient viral reservoir that sustains the infection 4,6–8 . Cytomegalovirus (CMV) vectors were designed to exploit this putative window of vulnerability, based on their ability to elicit and indefinitely maintain high frequency, effector-differentiated, and broadly targeted virus-specific T cells in potential sites of early viral replication 5,9,10 . Indeed, the pattern of protection observed in approximately 50% of RhCMV/SIV vector-vaccinated RM after intrarectal SIVmac239 challenge was consistent with early immunological interception of the nascent SIV infection at the portal of viral entry and immune control before irreversible systemic spread 5 . Protected RM manifested a very transient viraemia at the onset of infection, followed by control of plasma SIV levels to below the threshold levels of quantification, except for occasional plasma viral ‘blips’ that waned over time, and after one year, demonstrated only trace levels of tissue-associated SIV RNA and DNA at necropsy using ultrasensitive assays. The occurrence of plasma viral blips and the recurrence of ‘breakthrough’ progressive SIV infection in 1 of the 13 RhCMV/ SIV vector-protected RM at day 77 after infection indicated that SIV was not immediately cleared from these protected RM, but the failure to find more than trace levels of SIV nucleic acid in systemic lymphoid tissues was consistent with the productive infection being largely contained at the portal of entry with the possibility of eventual clearance. Given the crucial importance of understanding the degree to which a highly pathogenic lentivirus can be contained or even cleared by adaptive immunity, we sought to define more precisely the spread and dynamics of SIV infection in RM that controlled the infection as a consequence of RhCMV/ SIV vector vaccination, and in particular, the extent to which residual SIV was eventually cleared from these animals. To establish the extent of SIV spread early after the onset of RhCMV/ SIV vector-mediated control, we studied a group of five RM vaccinated with RhCMV vectors containing SIV Gag, Rev/Tat/Nef, Env and Pol (but not Vif) inserts that were taken to necropsy within 24 days of controlling plasma viraemia after intrarectal inoculation with SIVmac239. All of these RM had measureable SIV RNA in plasma for one or two weekly time points after challenge, followed by at least three consecutive weekly samples with plasma SIV RNA below 30 copy equivalents (equiv.) per

Mitochondrial Participation in the Intracellular Ca2+ Network

Abstract: Calcium can activate mitochondrial metabolism, and the possibility that mitochondrial Ca2+ uptake and extrusion modulate free cytosolic [Ca2+] (Cac) now has renewed interest. We use whole-cell and perforated patch clamp methods together with rapid local perfusion to introduce probes and inhibitors to rat chromaffin cells, to evoke Ca2+ entry, and to monitor Ca2+-activated currents that report near-surface [Ca2+]. We show that rapid recovery from elevations of Cac requires both the mitochondrial Ca2+ uniporter and the mitochondrial energization that drives Ca2+ uptake through it. Applying imaging and single-cell photometric methods, we find that the probe rhod-2 selectively localizes to mitochondria and uses its responses to quantify mitochondrial free [Ca2+] (Cam). The indicated resting Cam of 100–200 nM is similar to the resting Cac reported by the probes indo-1 and Calcium Green, or its dextran conjugate in the cytoplasm. Simultaneous monitoring of Cam and Cac at high temporal resolution shows that, although Cam increases less than Cac, mitochondrial sequestration of Ca2+ is fast and has high capacity. We find that mitochondrial Ca2+ uptake limits the rise and underlies the rapid decay of Cac excursions produced by Ca2+ entry or by mobilization of reticular stores. We also find that subsequent export of Ca2+ from mitochondria, seen as declining Cam, prolongs complete Cac recovery and that suppressing export of Ca2+, by inhibition of the mitochondrial Na+/ Ca2+ exchanger, reversibly hastens final recovery of Cac. We conclude that mitochondria are active participants in cellular Ca2+ signaling, whose unique role is determined by their ability to rapidly accumulate and then release large quantities of Ca2+.

Cytotoxic-T-cell responses, viral load, and disease progression in early human immunodeficiency virus type 1 infection.

Abstract: Background Early in human immunodeficiency virus type 1 (HIV-1) infection there is a decline in viral replication that has been attributed to host immunity, but the components of this response, particularly the ability of cytotoxic T lymphocytes to control viral burden and influence the outcome of disease, are poorly understood. Methods We prospectively studied 33 patients with primary HIV-1 infection for HIV-specific activated cytotoxic T lymphocytes and memory cytotoxic T lymphocytes and compared these lymphocyte responses with changes in viral load and clinical status over the subsequent 18 to 24 months. Results Soon after infection, activated HIV-specific cytotoxic T lymphocytes, mediated primarily by CD8+ cells, were detected in 17 of 23 patients (74 percent). Memory cytotoxic T lymphocytes were found in 6 of 6 patients tested (100 percent) during the first three months of infection and in 17 of 21 patients (81 percent) tested during the first six months. The frequencies of memory cytotoxic T lymphoc...