However, when Sema3E and VEGF were added together, almost no migr

However, when Sema3E and VEGF were added together, almost no migration was observed (Figures 4K and 4L), indicating that Sema3E inhibits VEGF-induced migration. Moreover, Sema3E also blocked the basal level of migration (Figure 4I). Together, these results support the idea that Sema3E can dominantly block the attractive effects exerted upon Plexin-D1-expressing endothelial cells. The appealing hypothesis that Protein Tyrosine Kinase inhibitor emerged, based on these in vitro results, is that Sema3E acts as a repulsive guidance cue for both trigeminal axons and blood vessels and serves to organize the double ring neurovascular structure

surrounding the follicle. If this hypothesis is true, what accounts for the relative position of the nerves and vessels, with the nerve ring consistently positioned inside of the vessel ring? In particular, it is puzzling how trigeminal axons expressing Plexin-D1 are able to innervate an area that is so close to a secreted repulsive cue. One potential possibility is that the abundance of Plxnd1 mRNA transcripts in the

TG may not reflect protein levels at the nerve terminal. To test this idea, we performed AP-Sema3E binding on tissue sections to detect Plexin-D1 protein along the trigeminal nerve ( Chauvet et al., 2007 and Gu et al., 2005). As shown in Figure 5A, although Plexin-D1 protein is highly expressed in the trigeminal nerves projecting to the whisker follicle, surprisingly, Plexin-D1 protein is very low in the nerve terminals (white arrowheads Tyrosine Kinase Inhibitor Library order in Figures 5B and 5C). This difference is not due to our inability to detect protein binding at the nerve terminal, because Npn-1 protein is equally well detected in both the projecting axons and the nerve terminal by AP-Sema3A binding (arrowheads in Figures 5D and 5E). The absence of AP-Sema3E binding on the vessel ring (red arrow in Figure S3C) of Plxnd1 null mice further next confirmed that Plexin-D1 is the only receptor for Sema3E in the vessel ring ( Figures S3A–S3D). The absence of AP-Sema3E binding on the nerve ring in Sema3e

null mice also ruled out the possibility that the lack of AP-Sema3E binding may be due to the sequestration of Plexin-D1 by endogenous Sema3E ( Figures S3E–S3H). To further confirm the selective downregulation of Plexin-D1 protein in the TG nerve terminals, we also performed anti-Plexin-D1 immunohistochemistry ( Chauvet et al., 2007). Consistent with the AP-Sema3E binding result, TG nerve terminal exhibited extremely low Plexin-D1 immunoreactivity ( Figures 5F and 5G). Therefore, the Plexin-D1 protein is selectively downregulated in the TG nerve terminals, which enables the nerves to innervate areas close to the Sema3E-expressing region and form the inner ring. In stark contrast to the low expression of Plexin-D1 in the nerve ring, the Plexin-D1 protein level visualized by both AP-Sema3E and anti-Plexin-D1 antibodies in the blood vessels is remarkably high (white arrows in Figures 5B, 5C, 5H, and 5I).

For example, while the monkey performs a visual discrimination ta

For example, while the monkey performs a visual discrimination task, the noradrenergic neurons in the LC exhibit both phasic and tonic modes of firing, which are correlated with good and bad performance (Usher et al., 1999). Subsequent experiments showed that the phasic activity of LC neurons occurs specifically before the behavioral response, and it may serve to facilitate the task-related decision process (Clayton et al., 2004). In a study in the rat performing an odor-guided decision task, serotonergic neurons in the DRN showed transient firing precisely time locked to a variety of

task-related events (Ranade and Mainen, 2009). Another study in the monkey showed that firing rates Doxorubicin in vitro of the DRN neurons were modulated by both the expected and received reward sizes (Nakamura et al., 2008). Neurons in the primate basal forebrain are also modulated by novel or reinforced stimuli (Wilson and Rolls, 1990). In behaving rats, the noncholinergic basal forebrain neurons showed strong burst responses to

both reward- and punishment-predicting stimuli, and the occurrence of the burst is strongly correlated with successful sensory detection (Lin and Nicolelis, 2008). The neuromodulator especially linked to vigilance and attention is ACh. In the rat, behaviorally relevant sensory cues can evoke transient increases in ACh concentration in the prefrontal cortex at the time scale of seconds (Parikh et al., 2007), and activating cholinergic transmission in the prefrontal cortex improves the performance of a sustained attention task (St Peters et al., 2011). LDN-193189 clinical trial A recent study based on genetic manipulation with recombinant viral vectors in the prefrontal cortex further demonstrated the importance of nicotinic ACh receptors (nAChRs) in sustained attention

(Guillem et al., 2011). Cholinergic signaling is also involved in selective attention. In the monkey performing a top-down spatial attention task, local application of ACh in the primary visual cortex was found to enhance the attentional modulation of neuronal firing rates, whereas mAChR Mephenoxalone antagonist had the opposite effect (Herrero et al., 2008). Together, these studies indicate that in addition to the daily sleep-wake cycle, the subcortical neuromodulatory circuits also serve to regulate arousal and attention on a faster time scale. Numerous studies in monkeys performing selective attention tasks have shown increased neuronal responses (Reynolds and Chelazzi, 2004), which are thought to enhance the perceptual saliency of the attended stimuli. Recent studies have shown that attention also causes a decrease in stimulus-independent correlated firing between neurons (Cohen and Maunsell, 2009; Mitchell et al., 2009), which may improve sensory encoding at the ensemble level (Zohary et al., 1994).

We focused on the genes that are misregulated in Bhlhb5 mutant mi

We focused on the genes that are misregulated in Bhlhb5 mutant mice at the time of the axon targeting defects (i.e., from E13.5 to E17.5). One of these genes is Cdh11, a classic type II cadherin that mediates homophilic cell-cell adhesion ( Kimura et al., 1995). Cdh11 mRNA is expressed in differentiating neurons of the cortical plate, including layer V projection neurons that form the corticospinal tract ( Kimura

et al., 1996). Furthermore, we found that Cdh11 protein is highly expressed in the axons of corticofugal neurons at E16.5, when these projection neurons are extending their projections through the internal capsule ( Figure 7C, white arrows), consistent with the idea that Cdh11 selleck inhibitor may play a role in their guidance. In addition, the subcortical expression pattern of Cdh11 is

suggestive of a possible role in regulating the connectivity of corticospinal motor neurons. In particular, Cdh11 is specifically expressed in a number of intermediate subcortical targets where corticospinal motor neurons form collaterals, namely the red nucleus, the basilar pons, and the inferior olive ( Kimura et al., 1996). Importantly, Bhlhb5 and this website Prdm8 bind to an intron within the Cdh11 gene ( Figures 5D, 5G, and 5J) and Cdh11 mRNA is upregulated in both Bhlhb5 and Prdm8 mutant mice during embryonic development ( Figures 7A and 7B). Upon loss of Bhlhb5, the overall level of Cdh11 protein appears elevated, whereas the pattern of Cdh11 expression is unaffected ( Figures 7C and 7D), suggesting that a Bhlhb5/Prmd8 repressor complex may function to restrain the level of Cdh11 rather than its distribution. Based on these observations, we hypothesized that Cdh11 might be a target of the Bhlhb5/Prdm8 repressor complex whose upregulation in the absence of Bhlhb5 or Prdm8 leads to a disruption of the formation of the corticospinal science tract. Specifically,

overexpression of Cdh11 in axons of corticospinal motor neurons might impede their progress due to enhanced adhesion to Cdh11-expressing intermediate targets. This might then prevent these Cdh11-overexpressing axons from extending past Cdh11-expressing intermediate targets and into the spinal cord. If so, we reasoned that reducing the level of Cdh11 in Bhlhb5 mutant mice might at least partially rescue the axon extension defects in corticospinal motor neurons. To test this idea, we obtained Cdh11 mutant mice (Cdh11−/−), which lack functional Cdh11 due to a targeted disruption of the extracellular domain and most of the transmembrane domain ( Horikawa et al., 1999). Importantly, mice lacking Cdh11 show normal targeting of corticospinal axons ( Figure 7G; data not shown).

Timothy McKinsey for generously sharing the P-S259 class IIa HDAC

Timothy McKinsey for generously sharing the P-S259 class IIa HDAC antibody, and Dr. James Bibb (UTSW) for advice on in vitro Cdk5 assays. We also acknowledge Dr. Eric Olson (UTSW) for sharing HDAC5 KO mice and Yong-Chao Ma (CMRC/Northwestern) for critical reading of the manuscript. L.N.S. was supported by fellowships from NIDA (T32 DA07290 and F32 DA027265) Volasertib in vitro and the FRAXA Foundation. C.W.C. acknowledges the generous support of the Whitehall Foundation, the Simons Foundation (SFARI grant), NIDA (DA008277 and

DA027664), and NEI (EY018207 and a research supplement for underrepresented minorities to M.B.C.). “
“Spontaneous neurotransmitter release is a salient feature of all presynaptic nerve terminals (Fatt and Katz, 1952). Recent studies have shown that these action potential (AP)-independent release events are essential regulators of synaptic homeostasis in terms of both presynaptic release rate and postsynaptic sensitivity (Aoto et al., Carfilzomib mw 2008, Frank et al., 2006, Lee et al., 2010, Sutton et al., 2006 and Sutton et al., 2007). Moreover, there is growing evidence that postsynaptic receptors and signaling elements that respond to spontaneous release events diverge from those that respond to evoked release (Atasoy et al., 2008,

Sara et al., 2011 and Sutton et al., 2007), suggesting a spatial segregation of the two forms of neurotransmission (Zenisek, 2008). Furthermore, a number of studies have provided evidence that presynaptic vesicle populations giving rise to spontaneous release are distinct Phosphoprotein phosphatase from those that

carry out AP-driven neurotransmission (Chung et al., 2010, Fredj and Burrone, 2009, Mathew et al., 2008, Sara et al., 2005 and Virmani et al., 2005). However, this notion remains controversial as some studies have provided contradictory results (Groemer and Klingauf, 2007, Hua et al., 2010 and Wilhelm et al., 2010). In the absence of molecular tags that identify a functionally distinct subpopulation of SVs, it is difficult to ascertain whether these observations disagree in substance or are merely due to vagaries of distinct experimental settings (Chung et al., 2010, Groemer and Klingauf, 2007, Prange and Murphy, 1999 and Sara et al., 2005). Lack of molecular insight into this putative functional heterogeneity also renders the examination of specific signaling consequences of spontaneous release independent of other forms of neurotransmission difficult (Kavalali et al., 2011 and Ramirez and Kavalali, 2011). Synaptobrevin2 (syb2), a key vesicular SNARE essential for all forms of neurotransmission in the CNS, is widely distributed among all vesicle pools as its absence gives rise to deficits in evoked and spontaneous neurotransmission (Schoch et al., 2001).

As shown previously with VSDI for electrical events, we now demon

As shown previously with VSDI for electrical events, we now demonstrate that Ca2+ waves propagate continuously through the cortex, recruiting large areas, perhaps even the entire cortex. In contrast to studies applying VSDI (Huang et al., 2010), we did not observe spiral or other nonlinear wave patterns. A possible explanation for this discrepancy may be that VSDI reflects primarily subthreshold activity, whereas Ca2+ imaging using fluorescent dyes mainly reflects suprathreshold neuronal activity (Garaschuk et al.,

2006b; Lütcke et al., 2010; Rochefort et al., 2009). The first field potential recordings of thalamic slow-wave oscillations were obtained in hemidecorticated cats in vivo (Timofeev and Steriade, 1996). In that study, the authors provided evidence from a small sample of combined cortical EEG and thalamic

Vemurafenib cost field potential recordings that spontaneous cortical waves preceded the associated thalamic ones. In the present study, we determined the corticothalamic wave latencies only for sensory- and optogenetically evoked waves, because these have, unlike spontaneous waves, a defined, unique site of cortical initiation. For such evoked waves, we demonstrate a clear temporal dominance of cortical over the thalamic wave initiation. Thus, visually evoked Ca2+ waves as well as Ca2+ waves evoked by intrathalamic optogenetic stimulation occur first in the buy Y-27632 visual cortex and only after a delay of about 180–200 ms in the dLGN. We emphasize that our findings only apply to the slow-wave activity. The primary fast neuronal activation upon visual stimulation will take place in the visual thalamic nuclei first, before being transmitted to the thalamorecipient cortical layer 4. Irrespective of their mode of initiation, Ca2+ waves were found to be remarkably unitary with virtually constant amplitudes and durations at a given brain location. This suggests that during waves of different origins, including the spontaneous, sensory-evoked, or optogenetically induced ones, a similar number of neurons participates on average in the slow oscillatory activity.

Our results obtained using optical Ca2+ recordings reveal Liothyronine Sodium properties of the slow oscillatory events that were not recognized previously. First, we observed an all-or-none behavior of the Ca2+ waves. The analysis of the optogenetically evoked waves particularly demonstrated that light pulses as short as 3 ms either evoke a full wave or no wave at all. Similarly, different light intensities for a given duration of the stimulating light pulse either evoked a full wave or no wave at all. Second, repetitive stimulation allowed the induction of consecutive waves only for intervals that were longer than about 2.5 s. For shorter intervals, wave initiation was either partially or, for very short intervals, completely refractory.

Finally, astrocytes are increasingly becoming implicated in a var

Finally, astrocytes are increasingly becoming implicated in a variety of human diseases from leukodystrophies, congenital epilepsy syndromes, to neurodevelopmental

disorders, and beyond. Reactive astrocytes are a hallmark of nearly all major human CNS neurodegenerative conditions ( Zamanian et al., 2012). How do astrocytes do all this? Is there only one type of astrocyte? For many years, investigators have reported different morphologies of CNS astrocytes, but electrophysiological correlates have not been clearly demonstrated. Although astrocytes have traditionally been considered a homogeneous population of cells, steady reports of their increasingly diversified functional roles Ipatasertib nmr CCI-779 in vitro in mammals brings into question whether astrocyte subtypes may have been elaborated in complex brains to carry out enhanced regional functions. For example, expression profiling studies of astrocytes have generated databases suggesting heterogeneous

functions that may be organized according to brain region. Astrocyte cocultures from brain and spinal cord can show differential effects in regulation of neural stem cells, and indeed, SVZ stem cells, which bear similarities to astrocytes, have been shown to be heterogeneous in terms of their progeny output (Merkle et al., 2007). A new type of radial glia stem cell, the outer radial glia (oRG), appears to function in the mammalian brain to contribute further rounds of progeny production increasing brain size and complexity (Rowitch and Kriegstein, 2010). These findings are augmented by the notion that evolutionary pressure might be a driving force for diversified astrocyte functions, discussed further below. The diversity of functional roles continuously

carried out by glia make them indispensable GPX6 for CNS function. For example, glia have to balance neuronal requests for energy, maintain the concentration of multiple extracellular ions, secrete growth factors, survey the nervous system for injury, all while reading neuronal activity and taking part in some aspects of signaling. The complexity of glial functions raises multiple experimental obstacles. First, because glia do so much, they are indispensable in higher organisms and their manipulation often leads to neuronal demise and death of the organism. Second, it is challenging to measure any of these functions in vivo, never mind measuring them all at once.

, 2009) ( Figure 2A) The locomotion rate and motile fraction

, 2009) ( Figure 2A). The locomotion rate and motile fraction

of pdf-1; npr-1 and pdfr-1; npr-1 double mutants during the L4/A lethargus were significantly lower than in npr-1 single mutants ( Figures 3D–3F). Inactivating PDF-1 and PDFR-1 had a much less dramatic effect on adult locomotion in pdf-1; npr-1 and pdfr-1; npr-1 double mutants ( Figure S3A). Thus, increased signaling by PDF-1 and PDFR-1 in npr-1 mutants was required for the increased motility during lethargus. The npr-1 foraging ZVADFMK defect was unaltered in pdf-1; npr-1 and pdfr-1; npr-1 double mutants ( Figure S3B), indicating that PDF was not required for other npr-1 phenotypes. Inactivating PDF-2 had little effect on the locomotion of npr-1 mutants during lethargus ( Figures S3C and S3D), indicating that PDF-1 is the major form of PDF involved in lethargus behavior. Collectively, these results suggest that PDF-1 functions as an arousal peptide in npr-1 mutants, preventing locomotion quiescence during lethargus. PDF-1’s effects on arousal were specific, because knockdown of BYL719 supplier 14 other neuropeptides expressed in the RMG circuit had no effect on the npr-1 lethargus defect ( Figure S3E). If PDF-1 functions as an arousal peptide, PDF-1 expression or secretion should be inhibited during lethargus, when animals are quiescent. We did several experiments to test this idea.

The abundance of pdf-1 and pdfr-1 mRNAs (assayed by quantitative PCR) was unaltered during the L4/A lethargus, whereas expression of mlt-10 (a gene required for molting) was significantly increased, as expected ( Figure S4A) ( Frand et al., 2005). To assay PDF-1 secretion, we expressed yellow-fluorescent-protein (YFP)-tagged proPDF-1 with the pdf-1 promoter ( Figures 4A and 4B). During DCV maturation, the YFP linked to proPDF-1

is cleaved by proprotein convertases and is subsequently secreted by DCV exocytosis. To assess the level of PDF-1 secretion, we analyzed PDF-1::YFP fluorescence in the endolysosomal compartment of coelomocytes, which are specialized scavenger cells that internalize proteins secreted into the body cavity ( Fares and Greenwald, 2001; Sieburth et al., 2007). either The PDF-1::YFP secretion reporter produced high levels of coelomocyte fluorescence in both L4 larvae and adults, whereas dramatically lower coelomocyte fluorescence was observed during the L4/A lethargus ( Figures 4A and 4B). Coelomocyte fluorescence produced by a second secretion probe (mCherry-tagged RIG-3 expressed in cholinergic neurons) ( Babu et al., 2011) was unaltered during lethargus ( Figure S4B), indicating that secretion and coelomocyte function were not globally inhibited during lethargus. If decreased PDF-1 secretion during lethargus is a cellular mechanism for inducing quiescence, we would expect that mutants retaining or lacking locomotion quiescence would exhibit reciprocal patterns of PDF-1 secretion during lethargus. We did several experiments to test this idea.

The work of this Laboratory is supported by the Wellcome Trust, L

The work of this Laboratory is supported by the Wellcome Trust, London. I am grateful to Carlo Cellucci, Marco Federighi, Tomohiro Ishizu, Konstantinos Moutoussis, and Dragan Rangelov for their comments on earlier versions of the manuscript. “
“Steve Fulvestrant price Heinemann, one of the fathers of modern molecular neuroscience and a pioneer in neurotransmitter receptor biology, passed

away on August 6, 2014, in La Jolla, California. Steve was well known in the neuroscience community as a genuinely nice guy with an unorthodox approach to research. For almost four decades in an extraordinarily productive career he helped to drive a quantum leap forward in elucidating the molecular components and cellular basis of excitatory neurotransmission in the mammalian CNS. These efforts paved the way for new insights into fundamental aspects of nervous system function, which led to advances in our understanding of neurological and psychiatric disorders. Stephen F. Heinemann Born in Boston in 1939, Stephen Fox Heinemann obtained a bachelor in science degree from the California Institute of Technology in 1962. He pursued his PhD in biochemistry at Harvard University in 1967 under the mentorship of Matthew Meselson, where

he studied the structure of DNA. His postdoctoral training

was carried out at the Massachusetts Institute of Technology and Stanford University School of Medicine, during Talazoparib ic50 which time he made contributions to the understanding of the genetics of the bacteriophage lambda life cycle. Soon after, his interest shifted to the nervous system, and he joined the Salk Institute for Biological Studies in 1970, where he became one of the founders of the Molecular Neurobiology Laboratory at the newly minted research institute that would become Edoxaban his second home. The Salk Institute, a masterpiece of Louis Kahn, one of the most influential architects of the 20th century, was designated a historical landmark in 1991, following his father’s “even a brick wants to be something” prophecy. A similar destiny was to be followed by the Molecular Neurobiology Laboratory, a program to be ranked number one in world neuroscience in the late 1980s, in part due to discoveries arising from the Heinemann laboratory. In his early days at the Salk Institute, Steve focused on the neurotransmitter receptors present at the neuromuscular junction. This was the model system widely used to understand synaptic transmission before brain synapses became tractable.

, 2010) it might prove difficult to differentiate the main drivin

, 2010) it might prove difficult to differentiate the main driving forces behind this observed phenomenon, i.e., colonic absorption window vs. a decreased gut wall metabolism in the colon, or both (Tannergren et al., 2009). To our knowledge however there is a paucity of studies investigating these bioavailability Libraries differences in a prospective manner. In addition, no attempts have been made to either elucidate the drug Sorafenib purchase and formulation properties associated

with the occurrence of such phenomenon or to correlate its magnitude to the aforementioned drug’s physicochemical, biopharmaceutical and biochemical properties. Due to the multifactorial nature of the problem, modelling and simulation (M&S), in particular

physiologically-based pharmacokinetic (PBPK) M&S, can be useful for the prospective analysis of the impact of such properties on the absorption and first past metabolism of CR formulations of CYP3A substrates. In silico PBPK models integrate current knowledge of both the system, i.e., morphophysiological factors (and their population characteristics) and drug properties that may influence oral drug absorption ( Jamei et al., 2009c). This approach has the advantage to allow the theoretical exploration of the interplay between the system and the drug properties and therefore hypothesize on the main GSK1120212 order driving forces that control drug absorption, transport and metabolism ( Darwich et al., 2010). Herein the relative bioavailability between CR and IR formulations of CYP3A substrates was investigated in order to understand how the physicochemical, biochemical and pharmaceutical properties of a drug (or drug product) can affect its oral bioavailability. Firstly, a literature survey was performed to collate clinical studies in which the pharmacokinetics unless of CYP3A4 substrates were

simultaneously investigated in both IR and CR formulations. Secondly, a systematic analysis was performed to investigate the impact that drug release characteristics and the drug-related physicochemical and biochemical properties defining oral bioavailability have on oral drug absorption and CYP3A4-mediated intestinal first pass metabolism. This was performed using in silico PBPK M&S. The aims of this study were to investigate possible mechanisms involved in the observed differences in oral bioavailability between IR and CR formulations by analysing the trends in fa, FG, and the systemic exposure (AUC). In addition, an attempt was made to identify the parameter space associated with the higher relative bioavailability of drugs formulated as CR compared to their IR counterparts and to correlate simulations with the observed clinical data gathered from the literature search. A literature survey was conducted using PubMed and Google Scholar in order to identify studies in which the pharmacokinetics of CYP3A4 substrates formulated as IR and CR was investigated.

In addition, children hospitalised with gastroenteritis were anal

In addition, children hospitalised with gastroenteritis were analysed to determine the risk factors associated Sorafenib mw with acute gastroenteritis mortality and prolonged hospitalisation. Hospitalisation for acute gastroenteritis: any hospitalisation of a child under five years of age with a primary or secondary attending-physician diagnosis of acute gastroenteritis. All hospital diagnoses had been coded using the ICD-9 classification of disease [11]. Multiple episodes of acute gastroenteritis in the same

child were included if the subsequent hospitalisation occurred more than two weeks after the previous hospitalisation. We excluded episodes of gastroenteritis in which the duration of diarrhoea exceeded 14 days at the time of admission, or which were coded as chronic diarrhoea episodes. Gestational age was categorised as preterm (<37

weeks gestation at birth) or term (≥37 weeks gestation at birth). Degree of dehydration was categorised by the attending physician into those who were ≤2.5% dehydrated, >2.5% but ≤5%, >5% but ≤7.5%, and >7.5% dehydrated. Dehydration of >5% was categorised as severe dehydration. Weight-for-age Z-scores for boys and girls from birth to five years (WHO child growth standards) were used to classify children as being malnourished. Those with weight-for-age less than minus two standard deviations were classified as being malnourished on admission. In those participants in whom a weight on admission was not available, malnutrition was considered present if the physician diagnosed KPT-330 mw kwashiorkor, marasmus or marasmic–kwashiorkor at admission. Descriptive diagnosis and diagnosis codes by hospital physicians were used to 3-mercaptopyruvate sulfurtransferase categorise participants as having a concomitant lower respiratory tract infection (LRTI) on admission. Patients with positive blood culture of a significant bacterial pathogen were defined as having bacteraemia.

Outcomes assessed were death during hospitalisation and duration of hospitalisation. Prolonged hospitalisation was defined as duration of hospitalisation greater than the median. Data were analysed using STATA version 11.0 (StataCorp, TX, USA). Incidence rates were calculated using the total number of acute gastroenteritis episodes during the study period and the total person years contributed by all those in the cohort. The censoring point was the date the participant turned five or death, whichever occurred first. Incidence rates stratified by HIV infection were not calculable by using person time analysis because we only imputed the HIV prevalence in the cohort and did not test all children. The imputed number of HIV-infected children was used as the Modulators denominators for cumulative incidence calculations when stratifying by HIV infection status. Hospitalised cases with an indeterminate or unknown HIV infection status were considered HIV-uninfected for the purposes of cumulative incidence calculations.