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To evaluate the effect of primary resistance and selected polymorphic amino-acid substitutions in HIV reverse transcriptase and protease on the CD4 cell count and viral load set point before the start of antiretroviral treatment.Design:Prospective cohort study.Methods:A total of 6180 individuals with a resistance test prior to starting antiretroviral treatment accessing care in HIV clinics across Europe who had at least one viral load and one CD4 test available were included in the analysis. The impact of amino-acid substitutions variants on viral load and CD4 trends was investigated using linear mixed models. Clusters of mutations were studied using principal component analysis.Results:Overall, the detection of any primary resistance was not associated with either the speed of CD4 cell decline or the viral load set point. However, transmitted nucleoside reverse transcriptase inhibitor and protease inhibitor resistance appeared to be weakly associated with lower viral load set points, as were the polymorphic G16E or Q92K protease mutations. There was some evidence suggesting that these effects varied according to HIV subtype, with the effects of transmitted nucleoside reverse transcriptase inhibitor and protease resistance being particularly marked among individuals with a subtype B virus. A cluster of five polymorphic protease substitutions at position 20, 13, 36, 69 and 89 was associated with less steep CD4 cell declines and lower viral load set points.Conclusion:Although we found little evidence for an association between primary resistance and CD4 speed of decline and viral load set point, the potential role of polymorphic protease (alone or in clusters) and their interplay with HIV subtype needs to be further evaluated. © 2018 Wolters Kluwer Health, Inc. All rights reserved.

GEM Amplifier Shaper Tracking ON Events (GASTONE) is a low-noise low-power mixed analog-digital ASIC designed to host 64 channels to readout the GEM inner tracker (IT) detector foreseen in the upgrade of the KLOE apparatus at the LNF e+e− DAFNE collider. Each channel is made of a charge sensitive preamplifier, a shaper, a discriminator and a monostable. Digital output data are transmitted via serial interface at 100 Mbit/s data rate. The chip has been developed by using the AMS CMOS 0.35 process. A 16 channels prototype has been produced and used to instrument the single layer IT prototype that has been tested with cosmic muons and a proton beam test at CERN.

Drug resistance is a major factor contributing to the failure of antiretroviral therapy. The rapid turnover of the human immunodeficiency virus type 1 (HIV-1) and the error-prone reverse transcriptase both contribute to the extensive emergence of resistant variants. The understanding of resistance and cross-resistance patterns is essential for the appropriate selection of treatment regimens for HIV-infected individuals. Although genotypic and phenotypic assays of drug resistance are not currently recommended for routine use, preliminary data has emphasized the role of resistance testing in guiding the choice of initial antiretroviral therapy, in explaining reasons for failure, and in selecting subsequent therapy for patients failing their current regimen. This article reviews the advantages and disadvantages of currently available resistance assays, as well as mechanisms and patterns of resistance and cross-resistance.

To compare the antiviral activity, safety and tolerability of didanosine dosed once and twice daily when administered in combination with stavudine dosed twice daily in human immunodeficiency virus type 1 (HIV-1)-infected individuals with little or no previous exposure to antiretroviral drugs.Comparative, multicentre, randomized, open-label, short-term study.Eighty-four HIV-1-infected adults with qualifying baseline CD4 cell counts of 200 to 500 cells/mm3 were included in the study. Of these, 43 patients received once daily didanosine plus twice daily stavudine (group A) and 41 subjects received twice daily didanosine plus twice daily stavudine (group B). The primary efficacy analysis used was the time-averaged difference (TAD) between treatment regimens of variations in plasma HIV-1 RNA levels from baseline over the first 12 weeks of therapy. Plasma HIV-1 RNA levels, CD4 cell counts and adverse events were monitored.At week 12, median HIV-1 RNA variations were -1.18 log10 copies/ml in group A and -0.88 log10 copies/ml in group B. For patients who were followed up to week 24, median variations of HIV-1 RNA levels from baseline were -1.21 log10 copies/ml in group A and -0.78 log10 copies/ml in group B. The TAD between the two treatment groups for variations from baseline plasma HIV-1 RNA levels over the first 12 weeks was 0.10 log10 copies/ml (95% confidence interval, -0.19 to 0.40), indicating equivalence.Once daily didanosine plus twice daily stavudine and twice daily didanosine plus twice daily stavudine are equally effective in reducing plasma HIV-1 RNA levels and increasing CD4 cell counts. Both regimens are safe and well tolerated.

Mycobacterium xenopi is one of the most common agents responsible for nontubercular mycobacterial pulmonary disease on AIDS patients. These lesions have been studied with conventional radiography, while CT has been used in patients with aspecific mycobacterioses or non-AIDS pulmonary conditions from Mycobacterium xenopi.We investigated the yield of CT in the study of lung lesions from Mycobacterium xenopi in AIDS patients.We examined 12 AIDS patients with pulmonary lesions from Mycobacterium xenopi, patients age ranged 30 to 46 years. All patients had CD4 blood levels lower than 250 cells/mL and Mycobacterium xenopi in the sputum. All patients underwent a standard chest radiograph and a CT examination. CT images were evaluated by three radiologists independently and the definitive diagnosis was made in the presence of a fourth radiologist.Chest CT showed parenchymal consolidation in 66% of cases, associated with bilateral basal bands in 16% of cases. Consolidation was unilateral in 41% of cases and most frequently involved the right lower lobe. Bilateral reticular interstitial involvement was seen in the patients (41%). Micronodules in 1 patient (8%) and mediastinal adenopathy in 33% of cases. Two patients had pre-xisting emphysema and 1 had bronchiectasis.The frequency of lung disease from Mycobacoerium xenopi has increased because of the spreading of the HIV infection. Such lung lesions in AIDS patients are aspecific in appearance and localization, which the clinical radiologist needs to consider to address treatment planning. The frequent finding of parenchymal consolidation and the absence of cavitary lesions may be referred to the poor capability of AIDS to produce an adequate inflammatory response. The lung lesions tend to distribute in the lower lobes unilaterally. Adenopathy was also a frequent finding. CT plays a fundamental role in studying the chest of these patients because it permits to locate lung lesions with higher accuracy than conventional radiography and to detect adenopathies, micronodules reticular interstitial involvement and bronchiectases.

Oral candidiasis frequently occurs in HIV-positive patients especially in those with advanced disease. To date, common anti-mycotic drugs are unable to prevent relapses and alternative therapy is necessary to reduce disabling effects. With the aim of verifying whether thymic hormone extract may be efficacious in these subjects, we enrolled 10 HIV-positive patients with recurrent and/or persistent oral candidiasis to be treated with thymopentin (oral inhalations). All patients benefited from the topical use of thymopentin, and in all cases there was marked increase in salivary secretory IgA which possibly accounted for the candidiasis improvement.

In order to evaluate the sensitivity of HCV test, Ortho Second and Third Generation HCV Elisa Tests were used in 315 sera collected from dialysis patients. The agreement between the two tests was 96.5%. RIBA Second and Third generation and PCR were tested in the eleven discordant sera and in specimens collected in 1992 from the same patients and in 20 Ortho-2 and Ortho-3 HCV negative patients. Ortho-3 test is capable of detecting anti-HCV seroconversion earlier than Ortho-2 with increased sensitivity and specificity, as is confirmed by RIBA and PCR tests.

Different genotypes of hepatitis C virus (HCV) have been shown to have distinct geographical distribution and to associate with variable clinical features. To evaluate their role in chronic hepatitis in Italian patients, we studied 495 consecutive cases with chronic hepatitis C seen in nine sentinel centres homogeneously distributed over Italy. HCV genotyping was carried out using a dot-blot hybridization assay with genotype-specific probes. Four hundred and eleven patients were viraemic and could be evaluated: 57% were found to be infected with HCV-1, 31% with HCV-2, 8% with HCV-3, 1% showed mixed infection and 3% were ascribed to HCV-2b or HCV-4 by direct sequencing. Geographical distribution showed discrete territorial variations. A history of drug addiction was commoner in patients infected with HCV-3. There were no significant differences in activity of liver disease among different HCV genotypes but the response to interferon therapy was reduced in patients infected with HCV-1 compared to HCV-2 or HCV-3.

There is currently no simple method to detect the antigen specificity of anti-HIV-1 IgG intrathecal synthesis (IS). Fifty-seven pairs of serum and corresponding CSF from 29 HIV-1 seropositive patients were adjusted to an identical concentration of total IgG and tested by a commercial HIV-1 Western Blot (WB) assay. IgG IS to a given HIV-1 protein was demonstrated when the corresponding band was present in CSF but absent or significantly less represented in serum. A total anti-HIV-1 IS was defined as the presence of an IS to one or more HIV-1 antigens. Our WB analysis of CSF and serum, compared with conventional mathematical formulas, showed a higher sensitivity in demonstrating anti-HIV-1 IgG IS. Moreover, the method disclosed which HIV-1 proteins represent the target of IgG IS. This procedure is easy to perform and therefore may represent a valuable tool to study central nervous system (CNS) involvement by HIV-1 during different stages of infection.

The long-term therapeutic efficacy of alpha IFN and the influence of preC variants on the type of response were evaluated in 25 patients with chronic hepatitis B, 14 HBeAg and 11 antiHBe positive patients, treated with alpha IFN and monitored for at least four years after discontinuing therapy. In both groups of patients, serum HBV-DNA became frequently undetectable by DNA dot blot during treatment, suggesting that alpha IFN has an antiviral effect both on HBeAg and antiHBe positive chronic carriers. However, long term follow up showed that the loss of viral DNA in antiHBe carriers was only transient, because all responder patients relapsed from 1 to 48 months after IFN withdrawal. In the HBeAg positive carriers, selection for preC mutants was observed at the end of follow up in 2 patients who seroconverted to antiHBe and remained viremic. Both the frequent occurrence of reactivations in antiHBe compared to HBeAg carriers, and the association of IFN therapy with preC mutant virus selection during long term post-treatment follow up observed in this study, indicate that preC variants are more resistant to IFN therapy than preC wild type HBV. Our data suggest therefore, that IFN therapy may be less frequently able to induce a permanent remission in patients infected with preC mutants.

We investigated a group of well characterized seronegative subjects "at risk" for HIV-1 infection including heterosexual partners of HIV-1 infected subjects and intravenous drug abusers. Pokeweed mitogen (PWM)-stimulated peripheral blood mononuclear cells (PBMCs) were studied for their ability to produce antibodies against HIV-1 structural proteins in vitro. Viral activity by means of HIV-1 isolation from PBMCs and presence of serum p24 antigen were also tested. In 7/42 cases (16.6%) HIV-1 immunoreactive specific antibodies were found, mostly directed against the envelope proteins (gp 120/160). Remarkably, none of these in vitro antibody producers yielded HIV-1 isolation in cell cultures or had detectable serum levels of p24 antigen.

We have evaluated a simple and sensitive culture technique for isolation of Human Immunodeficiency Virus Type-1 (HIV-1) from small amounts of whole blood. Data shown in the paper demonstrate that: 1) cell cultures from small amounts of heparinized whole blood (HWB) allow a high isolation rate in infected subjects at all stages of diseases; 2) among asymptomatic subjects the HIV-1 isolation rate is increased in cell cultures from HWB, with respect to cell cultures from peripheral blood mononuclear cells; 3) cultural results from HWB are not influenced by the presence of detectable serum p24 antigen, but a good correlation was found with the titre of anti p24 antibodies in serum; 4) continuous cell lines (such as Molt-3 cells) instead of peripheral blood mononuclear cells can be used, obtaining good results, for HIV-1 isolation from HWB; 5) frozen samples of HWB can be used in cell cultures for HIV-1 isolation; 6) the type of anticoagulant (Heparin or EDTA) used for the collection of blood does not influence viral replication in cell cultures from whole blood; 7) viral isolation from HWB is highly sensitive; amounts so small as five microliters of whole blood are sufficient, in some cases, to obtain viral replication in cell cultures; 8) the minimal dose of HWB sufficient to infect cell cultures (HWB M.D.I.) varied among different patients. Although this work failed to establish a correlation between this parameter and the clinical and immunological status of patients, it is conceivable that HWB M.D.I. could give information about viral load in blood and have a prognostic significance; 9) the HWB M.D.I. rise in patients treated with Zidovudine, suggesting that this method could be employed in the virological evaluation of trials with antiretroviral drugs.

The authors have studied the replicative kinetics and the induction of cytopathic effects of HIV-1 in primary co-cultures from infected subjects at various stages of the disease. Cultures from subjects with ARC or AIDS yielded HIV-1 replication more precociously and at higher levels compared to those from asymptomatic subjects; cytopathic effect "in vitro" were observed more frequently and earlier in cell cultures from ARC/AIDS subjects. Presented data indicate that the clinical and immunological deterioration during HIV-1 infection is related to viral replicative activity and suggest that the study of HIV-1 replicative kinetics in primary co-cultures may be helpful in predicting who will progress to AIDS.

Seven paired HIV-1 isolates from peripheral blood mononuclear cells (PMCs) and cerebrospinal fluid (CSF) of infected subjects at various stages of the disease were studied for their capacity to replicate in continuous cell lines (Molt-3 and U-937 cells) and to induce cytopathic effects "in vitro". Obtained results indicate that paired HIV-1 isolates from PMCs and CSF of the same patient can differ in their replicative activity "in vitro", suggesting that, at least in some cases, CSF isolates may represent a distinct subtype of HIV-1.

The authors present a simple and sensitive culture technique for isolation of Human Immunodeficiency Virus (HIV) from small amounts of heparinized whole blood. This method appeared to be highly sensitive at all stages of disease. Among asymptomatics HIV was isolated from 41.3% of subjects by using peripheral blood mononuclear cells in a standard co-cultivation technique and in 89.6% of cases using whole blood. In the group of AIDS patients, in contrast, HIV isolation rate of the two techniques was the same (94.4%). The presence of detectable p24 Ag in plasma did not affect cultural results and no false positive was observed in the control group. Although further studies are needed to better understand the biological significance of a positive cultural result obtained by this method, HIV isolation from whole blood can be routinely employed, especially when small amounts of blood are available.

A solid phase radioimmunoassay (RIA) procedure was developed and three methods for detection of IgM specific antibody to hepatitis A virus (anti-HAV IgM) were compared: triple antibody method, 2-MercaptoEthanol (2-ME) for IgM cleavage and Staphylococcal A Protein (StAP) for IgG absorption. Specificity and sensitivity of the tests were checked for evaluating acute and convalescent sera from 40 patients with serologically (seroconversion) diagnosed hepatitis A and 64 sera from patients with various acute viral diseases or with high titre of rheumatoid factor (RF). Specimens to be assayed for anti-HAV IgM were pretreated with 2-ME or StAP and tested by RIa using 125I labelled anti-HAV IgG. Triple antibody method showed to be more sensitive than other two methods giving false positive result in only one serum containing high levels of monoclonal RF. No significant difference in sensitivity and specificity was found between 2-ME and StAP procedure, but these methods were able to detect anti-HAV IgM for only two weeks after the onset of the disease, whereas triple antibody method gave positive results for at last seven weeks.

The contagiousness of hepatitis B virus (HBV) carriers with end-stage renal disease undergoing chronic hemodialysis has been ascribed to an immunologic tolerance for HBV antigens, especially hepatitis B core antigen, supporting persistently high levels of virus replication. In this context hepatitis B e antigen and core-associated DNA polymerase (DNA P) activity have proved to be distinct markers of HBV replication. In order to evaluate the potential infectivity of these subjects, thirty-five HBsAg positive hemodialysis patients were studied for the presence of HBeAg/anti-HBe system correlating the results with serum DNA P activity. Twenty out of 35 patients were HBeAg positive (57%) and 21 DNA P positive (60%). A highly significant correlation (P less than 0,001) was recorded between detection of HBeAg and presence of serum DNA P activity. These findings confirm that the majority of hemodialysis patients carrying HBsAg show high levels of virus replication so that the determination of HBeAg and DNA P activity other than HBsAg is required for the identification of patients highly infectious.

58 fecal specimens from 14 patients (10 hepatitis A, 2 hepatitis B and 2 infectious mononucleosis) were tested for the hepatitis A virus (HAV) and IgA anti-HAV by micro-solid-phase-radioimmunoassay. Only patients with hepatitis A were positive for HAV and/or IgA anti-HAV. In the first days of the disease we found HAV in the feces of 4 patients but it was never present after the sixth day. In all hepatitis A we found IgA anti-HAV in at least one fecal specimen and the titer of the antibodies increased in most cases during the course of the disease. The duration and the peak of the IgA response in the feces were strongly similar to other enterovirus infections. Some methodologic improvement both for HAV and IgA anti-HAV detection are suggested.

To clarify the biological, clinical and prognostic relevance of HIV isolation from plasma and explain the relation to p24 antigenemia, we studied these two markers in 67 anti-HIV positive subjects in different stages of the disease. The results suggest that discordances between these two parameters are not dependent on methodological problems and may be attributed to yet unexplained biological phenomena. Some hypotheses to explain the observed discrepancies are suggested.

One-hundred-and-two out of 788 consecutive patients (12.9%) hospitalized for an attack of acute hepatitis fulfilled criteria for non-A, non-B (NANB) hepatitis, and were followed prospectively for 8 to 70 months. Forty-one out of 97 patients showed a monophasic pattern of alanine aminotransferase (ALT), and all recovered completely. In contrast, 22 of 56 (39.2%) subjects with a polyphasic pattern of ALT exhibited persistent hypertransaminasemia for more than 14-18 months, and all of them developed chronic hepatitis. Thus, a polyphasic pattern of ALT seems to characterize one of two forms of NANB hepatitis, more frequently associated with parenteral exposure, absence of jaundice and a high tendency for the development of chronic hepatitis. This suggests distinct immunopathogenetic mechanisms from two unrelated NANB agents. Benign, non-progressive chronic persistent hepatitis is the prevalent form of chronic sequela of NANB hepatitis observed in our patients. Although many of these patients show a tendency to spontaneous remission of the disease, there is the possibility in some of a deterioration of inflammatory activity of the liver that has need of immunosuppressive therapy.

Fourteen out of 28 HBsAg/HBeAg-positive carriers with chronic persistent and active hepatitis were randomly assigned to human leukocyte interferon (a-IFN) treatment for three months. The remaining 14 patients served as controls. Each treated subject received a standard i.m. dose of 0.7-1.0 X 10(5)/kg/day reference units of a-IFN for 28 consecutive days, and then the same dose twice a week for two months. This treatment regimen was well tolerated, and no remarkable side effects were recorded. At six months the number of patients who permanently lost HBV-DNA from serum was significantly higher in the treated group (p = 0.006) than in the untreated group. These results suggest that a less expensive and well tolerated treatment regimen based on low dosage of a-IFN may be as effective in producing permanent inhibition of hepatitis B virus replication as a treatment regimen based on larger dosage.

ObjectivesTo evaluate the incidence and risk factors for liver enzyme elevations (LEE) in patients initiating first-line ART in the ICONA prospective observational cohort, between June 2009 and December 2017.Patients and methodsIn total, 6575 ART-naive patients were selected, initiating two NRTIs with the third drug being a boosted PI (n=2436; 37.0%), an NNRTI (n=2384; 36.3%) or an integrase strand transfer inhibitor (INSTI) (n=1755; 26.7%). HBV surface antigen and HCV RNA were detected in 3.9% and 5.8% of the study population. Inverse probability weighted Cox regression analysis was used to calculate the HRs, according to first-line regimen, for LEE, defined as ALT or AST increases of ≥2.5× upper limit of normal (ULN) for patients with normal baseline values or ≥2.5× baseline for patients with higher baseline values.ResultsOne hundred and eighty-three LEE occurred over 20722 patient-years of follow-up. After adjusting for the main confounders, the risk of LEE halved with INSTIs compared with NNRTIs (HR 0.46, 95% CI 0.25–0.86), with a significant reduction in the raltegravir group (HR 0.11, 95% CI 0.02–0.84 using the NNRTI class as reference). HRs for LEE were significantly higher in subjects with HBV or HCV coinfection, in patients with poorly controlled HIV infection and in those who acquired HIV through homosexual transmission.ConclusionsIn our study, INSTI use almost halved the risk of LEE compared with other regimens. This finding could be particularly important for choosing ART in patients with risk factors for liver toxicity such as HCV and HBV coinfections.

Background The aim of this study was to investigate the impact of anti-HBc (HBcAb) positivity on the progression of liver fibrosis (Fibrosis-4 score >3.25) in the Italian cohort of HIV-infected individuals naïve to antiretroviral treatment (ICONA). Methods All patients with FIB-4 Results Patients who were HBcAb+/HCV-/HBs antigen (HBsAg)- and HCV+/HBcAb+/HBsAg- or HBsAg+/HBcAb+/HCV- had CD4+ cell counts below the nadir and significantly higher prevalence of AIDS diagnosis at baseline than the other groups (P < .0001). A Cox regression model adjusted for age, HIV transmission mode, country of birth, and alcohol consumption showed a higher relative risk (HR) of progression to FIB-4 >3.25 in HCV+/HBcAb+/HBsAg- patients (HR, 7.2; 95% CI, 3 8–13.64). Conclusions HBcAb+ contributes to liver damage in HIV+/HCV+/HBcAb+/HBsAg- subjects. A careful monitoring for signs of previous HBV infection is needed in this kind of patients.

Objectives To describe: (i) factors associated with rapid and delayed ART initiation; (ii) rates of 12 week virological response; and (iii) virologically controlled retention in care by 1 year from ART initiation according to timing of start in a real-life setting. Methods All individuals in the Icona cohort diagnosed with HIV in 2016–17 who initiated ART were grouped according to the time between HIV diagnosis and ART initiation: Group 1, ≤7 days; Group 2, 8–14 days; Group 3, 15–30 days; Group 4, 31–120 days; and Group 5, >120 days. Multivariable logistic regression models were used to identify factors associated with: (i) the probability of rapid (Group 1) and very delayed (Group 5) ART initiation; (ii) the 12 week virological response (by a modified snapshot algorithm); and (iii) the probability of retention in care at 1 year (on ART with HIV-RNA Results A total of 1247 individuals were included [82 (6.6%) in Group 1, 115 (9.2%) in Group 2, 267 (21.4%) in Group 3, 641 (51.4%) in Group 4 and 142 (11.4%) in Group 5]. Main predictors of rapid ART start (Group 1) were low CD4 cell count and high HIV-RNA at first contact with the infectious diseases centre. There was no association between probability of virological response and timing of ART initiation. Overall, 90% of individuals remained on ART after 1 year, 91% with undetectable HIV-RNA. Participants of Italian nationality, those with higher CD4 cell count and lower HIV-RNA at ART initiation were more likely to be retained in care after 1 year. Conclusions In our high-income observational setting, we did not observe differences in the 1 year rate of virological response and retention in care according to timing of ART initiation.

Objectives We detail a procedure for generating a set of templates for the hippocampal region in Magnetic Resonance images, representative of the clinical conditions of the population under investigation. Methods The first step is robust standardization of the intensity scale of brain MR images, belonging to patients with different degrees of neuropathology (Alzheimer’s Disease). So similar tissues have similar intensities, even across images coming from different sources. After the automatic extraction of the hippocampal region from a large dataset of images, we address template generation, choosing by clusterization methods a small number of the extracted regions. Results We assess that template generation is largely independent on the clusterization method and on the number and the clinical condition of the patients. The templates are chosen as the most representative images in a population. The estimation of the ‘minimum’ number of templates for the hippocampal region can be proposed, using a metric based on the geometrical position of the extracted regions. Conclusions This study describes a simple and easily reproducible procedure to generate templates for the hippocampal region. It can be generalized and applied to other brain regions, which may be relevant for neuroimaging studies.

Purpose: The hippocampus, located in the medial temporal lobe (MTL), plays an essential role in learning and memory functions. Because of its frequent and early involvement in Alzheimer's disease (AD) and other neurodegenerative diseases, it is often targeted by both structural and functional imaging. Our aim is to increase the likelihood of early recognition and assessment of AD. Methods and Materials: We propose an approach that does not directly tackle the objective of hippocampal segmentation, but simply extracts from the right and left sides of a MR image two small fixed-size, parallelepiped-shaped sub images containing the hippocampi and adjacent structures (Hippocampal Boxes, HBs). We developed an automatic procedure for selecting an optimal number of HBs, starting from which we can extract both hippocampal formations in any MR image. We discriminate between controls and AD. This way MCI population can be evaluated and a prediction on the conversion to AD is made. Results: We extracted HBs from a set of 532 images from different sources, and developed a method to discriminate between converters and non converters to AD in a MCI population. The separation between Control and AD, measured as the area under the ROC curve, is 86,3%. For the Normal vs. Converted to AD cohorts, the area under the ROC curve is 88%. The forecast is checked against clinical follow up. Conclusion: The proposed approach consists in the possibility of automatically performing morphometric studies on the MTL. This procedure can quickly and reliably provide additional information in early AD diagnosis. The study has been established within the framework of the MAGIC-5 collaboration.