[7, 9, 10] check details The replication

of flavivirus generally occurs on virus-induced host cell membranes. DENV requires autophagy for efficient replication, with recent studies showing that DENV infection induces autophagy, and the inhibition of autophagy reduces significantly DENV replication and release of viral particles.[11-13] These structures may serve as a scaffold for anchoring the viral replication complexes, which consist of viral RNA, viral proteins and host cell factors.[14] Dengue is now considered an important neglected tropical disease. Although many studies have been carried out for almost a century, many aspects of disease remain unresolved. The great lack of knowledge on dengue pathogenesis is a major factor that contributes to a striking human and economic burden. Disease development is not fully understood, which has delayed the development of vaccines, treatments and effective methods for DENV detection.[15] After infection of an immune-susceptible host, an acute, self-limiting febrile systemic syndrome starts to develop. Resolution of infection normally occurs within 4–7 days and is associated with a robust innate and adaptive immune response. The diagnosis is largely clinical, treatment is supportive and disease control is limited to the elimination of its vectors.[1, 2] Primary infection in older children

and adults normally lead to DF, a febrile

illness accompanied by a combination selleck compound of non-specific symptoms that may include headache, retro-orbital pain, myalgia and occasionally haemorrhagic manifestations.[1, 16] Some patients, such as newborns and elderly people, occasionally develop DHF, the most severe form of dengue disease. The hallmark of DHF is the presence of plasma leakage and haemoconcentration, which can lead to the loss of intravascular volume and circulatory insufficiency.[16] Significant bleeding is also a clinical feature associated with severe disease. Bleeding can be observed in both DF and DHF; more severe bleeding, such as bleeding from the gastrointestinal tract, is found more frequently in DHF than in DF. Increased liver enzymes [aspartate aminotransferase/alanine aminotransferase (AST/ALT)] Glycogen branching enzyme and thrombocytopenia (platelet count < 100 000 cells/mm3) are commonly observed in both DF and DHF patients but are more severe in DHF.[16, 17] However, haematocrit readings can be affected by factors such as fever, dehydration and haemorrhage. Patients with DHF who have narrow pulse pressure (<20 mmHg) or who show signs of shock are classified as having DSS. Other severe clinical manifestations including hepatic failure and encephalopathy have been reported in dengue patients.[16-18] Viral load is controlled by the host after a few days, when signs of systemic inflammation are still observed.

The reasons for these divergent results are still unknown but as we understand more about the immune system in adipose tissue one could speculate several explanations for these discrepancies. One possibility is that microbiome differences between laboratories and between wild-type and knockout mice contribute to the difference in weight gain, as the microbiome has been to shown to significantly impact metabolism DAPT in vivo and the development of obesity,[65] as well as iNKT cell development.[66] We have exchanged cages between non-littermate wild-type and iNKT knockout mice to reduce the impact of the microbiome. However, the reference standard is to use wild-type and knockout littermates to eliminate

the impact of the microbiome, which were used in some,[63, 67] but not most, of the studies summarized above. Another plausible explanation is the age of mice

in each study. In young mice, there is a substantial population of iNKT cells and fewer regulatory T cells in adipose tissue, and at 8–16 weeks, iNKT cells accumulate further but decline in old age, whereas adipose regulatory T cells greatly accumulate in old mice.[51] Therefore, it is plausible that iNKT cells may be more influential in younger mice, whereas in older mice it is the regulatory T cells that dominate and the role of iNKT cells, or lack of them may be less dominant. It is also possible that for some reason both wild-type and iNKT-deficient animal Reverse transcriptase colonies in different laboratories have a more Th1 or more Th2 bias among iNKT cells or other lymphocytes, or in some colonies, there is a compensatory Selleck Neratinib mechanism when iNKT cells are absent from birth. Despite the divergent results using iNKT-deficient mice, other methods

to measure the effects of iNKT cells on obesity and metabolism are more consistent. First, over 14 independent studies have shown that iNKT cells (when measured accurately) are depleted in obesity, and all human studies have also found iNKT deficiency associated with obesity. Other immune cells that are shown to be protective in obesity,[52] such as regulatory T cells,[51] alternatively activated macrophages and eosinophils,[54] are depleted in obesity, whereas those that are shown to be pathogenic in obesity like CD8+ T cells[50] and classically activated macrophages[56] are increased in obese adipose tissue. Based on this comparison, which is not direct evidence and merely an association, iNKT cells appear to be part of the protective anti-inflammatory immune cell group that are lost in obesity as an inflammatory response takes over. More direct evidence comes from gain of function experiments, when iNKT cells are adoptively transferred into wild-type or iNKT-deficient obese mice or activated in wild-type obese mice. The majority of studies have shown that this has a positive impact on metabolic control and on protection against weight gain.

The means of sensitization is clinically relevant; but it is unlikely that the amount of pollen extract inhaled or instilled is quantitatively related to the strength of the reaction. In fact, instillation of a total amount of 16.6 μg in 33.2 μL of PBS of this allergen in five divided doses in one day into each of

eight mice induced a significant increase in the serum concentrations JAK drugs of nonspecific IgE Ab on day 14 in only one mouse (Ogita-Nakanishi et al., unpublished data). In contrast, an injection of the allergen into the area surrounding the nostrils (100 μg in 0.15 mL of PBS) resulted in an increase (≈ 10-fold of control) in serum IgE Ab production on day 10 (Fig. 4; references 7 and 8). Therefore, in the present study, we injected i.n. cedar pollen without adjuvant once into BALB/c mice to induce the initial stage of allergic rhinitis in various lymphoid organs, including the submandibular lymph nodes. The histology of the palates, cell yields from the NALT, and their phenotypic composition (Fig.

1) were essentially the same as those reported previously (17, 18). However, the total cell numbers in the NALT did not change significantly on days 0–14 after i.n. injection of the allergen; and the bulk cells did not produce significant amounts of IgE on days 0–14 (Figs. 2 and 3). Consistently, submandibular lymph nodes, but not the NALT, were clearly stained with i.n. injected Evans blue (Fig. 3, inset), suggesting that the NALT might Inhibitor Library supplier not drain extracellular fluid containing i.n. injected allergen. Alternatively, it has been shown that i.n. immunization with a single dose of 1 μL of PBS solution

containing pathogens into each nostril can establish effective immunity against pneumococci, group A streptococci, influenza virus, Bordetella pertussis, herpes simplex virus or Streptococcus mutans in mice (18, 23–28). These results suggest that the once only application of pathogens in 1 μL of PBS solution into each nostril is sufficient to reach both non-NALT lymphocytes and NALT lymphocytes. In contrast, application of even five times as much cedar pollen (3.32 μg) in 6.64 μL of PBS solution into each nostril might be insufficient Alanine-glyoxylate transaminase to elicit or penetrate into the NALT or non-NALT lymphoid tissues (Ogita-Nakanishi et al., unpublished data). Previously, we reported that wild-type, IFN-γ -/-, but not IL-4 -/-, mice sensitized once (i.n. or i.p.) or twice (s.c. or i.v. and s.c.) showed a significant increase in nonspecific IgE Ab in their serum (8). In order to determine which population of PBMCs was involved in the in vitro production of nonspecific IgE Ab in that study, we separated PBMCs from mice sensitized s.c. once into three cell populations (i.e., monocytes, lymphocytes, and granulocytes) by Percoll density-gradient centrifugation. (i) The lymphocyte- or monocyte-rich fraction alone does not produce of IL-4 and IgE Ab.

E. coli strains were grown in LB medium or TSB (BD Diagnostic Systems, Sparks, MD, USA). Construction of a crp deletion mutant of J29 was performed by the methods of Donnenberg and Kaper (37). In short, the crp gene was amplified by PCR with E. coli J29 as the template. The amplified fragment was cloned into the BamH I and Sal

I sites of pMW119. A 351-base pair internal deletion of crp gene was created by digestion with Hinc II (Toyobo Life Science, Tokyo, Japan) and ligation with T4 DNA ligase (Boehringer Mannheim, Burlington, ON, Canada) according to the manufacture’s recommendations. The internally deleted gene was subcloned into pCVD442 (37), and the resulting IWR-1 solubility dmso plasmid transformed into E. coli SM10λpir (38) by electroporation followed by selection with ampicillin. This recombinant plasmid was transferred from E. coli SM10λpir into a nalidixic resistant clone of E. coli J29 by filter mating followed by selection with nalidixic acid and ampicillin. Plasmid excision events were identified by selection for sucrose resistance followed by screening for ampicillin and kanamycin susceptibility, which is indicative of loss of suicide vector sequences. Deletion of the chromosomal crp gene was confirmed by PCR screening. The primer sets and PCR conditions have been described previously (36). One of the resulting mutant strains was designated AESN1331; the mutant strain was cultured in TSB and stored

as a ABT-263 mw frozen culture (-80°C) in 50% glycerol. Fertilized eggs and chickens of SPF white leghorns of the

line M were obtained from the Laboratory Animal Research Station, Nippon Institute for Biological Science (Yamanashi, Japan). The eggs were Sirolimus order incubated at 37–38°C in a relative humidity of approximately 55%. Animal utilization protocols were approved under the guidelines of Nippon Institute for Biological Science on Animal Care. The presence of the O78 surface antigen was established by slide agglutination with the corresponding antiserum (Denka Seiken, Tokyo, Japan). Colony diameter was tested by culturing bacteria on trypticase soy agar (BD Diagnostic Systems) for 24 hrs at 37°C and then measuring the diameters of three separate colonies with a ruler (1 mm resolution). Colony color was assessed following culturing on MacConkey agar (BD Diagnostic Systems for 24 hrs at 37°C. Biotyping was performed with the API20E bacterial identification system (bioMerieux sa, Marcy l’Etoile, France). For assay of hemolytic activity, blood agar plates containing 5% sheep blood in LB medium were streaked with over-night cultures and examined for clear zones of erythrocyte lysis after 20 hrs incubation at 37°C (36). Adsorption of Congo red was tested by the method of Corbett et al. (39). Detection of the following genes was performed by PCR: papC, which encodes P fimbriae; tsh, which encodes temperature-sensitive hemagglutinin; cvaC, which encodes colicin V, and iss, which encodes increased serum survival protein.

To the best of our knowledge, the former mutation (A1017T) has not previously been reported. To make a clinical diagnosis of NPC is often difficult, as in the present case, due to the extreme clinical heterogeneity of the disease: there is a wide range in the age of onset (ranging from the perinatal period to late adulthood), survival time (ranging from days to more than 60 years), and initial manifestations

(including hepatic, pulmonary, neurological and psychiatric abnormalities).[2, 5] This diversity of clinical presentation may cause significant diagnostic delay.[5, 12-14] The absence of organomegaly in the present patient caused further difficulties for assignment of a clinical diagnosis Selleck SCH727965 of NPC; only 10% of juvenile-onset, but 50% of adult-onset, NPC patients lack hepatosplenomegaly.[2, 5] However, when we retrospectively reviewed the clinical features of this patient, we could have considered the possibility of NPC, based on the concurrence of childhood-onset ataxia and vertical supranuclear ophthalmoplegia. Early diagnosis is important, since miglustat has proven to be effective for treatment of progressive neurological changes in NPC patients.[2] Predominantly frontotemporal atrophy was a unique feature of the present

case. Some investigators have previously reported frontal Ku-0059436 cost atrophy in some NPC cases as evidenced by clinical imaging. MRI and positron emission tomography have revealed frontal lobe atrophy in some patients, especially in those with predominant psychiatric or cognitive symptoms.[5, 14-16] Other investigators have reported pathologically confirmed frontal lobe atrophy in NPC cases.[3, 17] Klünemann et al. reported an autopsy case of adult-onset NPC due to a mutation of HE1/NPC2, exhibiting frontal lobe atrophy and lysosomal storage virtually restricted to neurons.[17] Histopathological analysis has previously revealed

that NFTs were more intensely distributed in the frontal lobe than in the occipital lobe in NPC,[3] suggesting that the disease process predominantly affected the frontal brain areas. Although an MRI volumetric study has revealed partial reductions in the temporal lobe gray matter volume, such as of the planum temporale, Heschl gyrus, hippocampus and parahippocampal gyrus,[18] involvement Vorinostat datasheet of the entire temporal lobe in NPC has not previously been described, to our knowledge. Involvement of almost the entire temporal lobe, as in the present case, may be a manifestation of the end-stage of the disease course. The formation of LBs in various cortical regions and brainstem nuclei is another conspicuous feature of the present patient, which supports the previously reported notion of NPC as an α-synucleinopathy.[6] The interactions between tau and α-synuclein may promote their assembly, as has been suggested.

Moreover, our results offered a mechanistic explanation for pre-BCR autoreactivity by suggesting recognition and binding between neighboring pre-BCR molecules. Here, we investigate the hypothesis that autoreactivity is critically required for the positive selection of precursor B cells in vivo and that the central role of the pre-BCR

is the initiation of selection signals that can be replaced by signals from autoreactive PLX4032 chemical structure BCRs. Based on our observations on the functional similarity between pre-BCRs and self-reactive BCRs in vitro, we hypothesized that if the pre-BCR was a specialized autoreactive receptor, then expressing an autoreactive BCR should overcome the developmental block in pre-BCR-deficient mice. To test this, we crossed 3-83Igi mice, in which the HC (3-83Hi) and LC (3-83κi) variable gene segments of the autoreactive BCR 3-83 are knocked into the IgH and Igκ loci respectively, with λ5-deficient mice 6, 10, 13. The 3-8 3 BCR recognizes MHC class I proteins of different haplotypes with different affinities, with H-2Kb being strongly recognized, whereas the binding affinity to H-2Kd ranked as the lowest 14–16. Thus, the 3-83 BCR is strongly autoreactive on the H-2b background and should rescue pre-BCR deficiency when Fulvestrant molecular weight combined with

H-2b but not with H-2d. Indeed, flow cytometric analysis of bone marrow cells showed that autoreactive B cells (3-83Hi/3-83κi on the H-2b background) overcame the early developmental block in λ5-deficient mice (Figs. 1A and B, S1A). In contrast, Thymidylate synthase on the H-2d background lacking the specific auto-antigen, the 3-83 BCR failed to efficiently rescue B-cell development. The majority of the B lineage

cells in the bone marrow were pro-B cells, which, similar to λ5-deficient cells bearing WT Ig genes, express the early marker CD43 (Fig. 1A and B). In agreement with the rescue of B-cell development in the bone marrow, λ5-deficient mice expressing the 3-83 BCR on the H-2b background showed normal proportions of B cells in the spleen and restored B-cell numbers. On the H-2d background, however, B-cell numbers were significantly reduced, suggesting that 3-83 BCR expression alone is not sufficient to rescue B-cell development (Fig. 1C–E). Together, the above results suggest that an autoreactive BCR efficiently initiates B-cell development and rescues an otherwise severe developmental block caused by pre-BCR deficiency. To further investigate the ability of autoreactive BCRs to drive early B-cell development, we injected HSCs from λ5-deficient 3-83Hi/3-83κi mice into immune deficient Rag-2/γC−/− mice 17. The cells were mixed in various proportions with WT HSCs to test the capacity of autoreactive B cells to compete with WT cells (Fig. 2A).

These inflammatory processes take place in the synovial membrane [4], and are characterized by lymphocyte

and macrophage invasion [5, 6] and elevated proinflammatory cytokines [7]. Because there are currently no therapeutic approaches to halt OA progression, much hope has been expressed regarding the development of new therapeutic strategies, including cell-based approaches. In this context, mesenchymal stem or stromal cells (MSCs) have been investigated extensively throughout the past two decades mainly for their regenerative potential [8-10]. Their immunosuppressive competence has, however, become another important field of research (overview in [11] and [12]). Therefore, MSCs have been investigated in animal ABT-737 mouse models of multiple sclerosis [13], pulmonary fibrosis [14], renal failure [15] and myocardial infarction [16]. In a clinical setting, MSCs have been used successfully as an immunosuppressive treatment in patients with severe graft-versus-host disease [17]. MSCs were also identified to play a crucial role in modulating the inflammatory processes in rheumatoid arthritis [18]. In an selleck chemical animal model of collagen-induced arthritis, MSCs reduced inflammation significantly in

the joints by reducing proliferation and modulating cytokine expression [19]. The mechanisms of MSC-mediated immunosuppression are unclear and still controversial [20, 21], while representing a promising target of cell-based therapies in diseases with important inflammatory processes. MSCs have been proved to suppress T cell proliferation successfully both in vitro and in vivo [22, 23]. Recent studies have also shown that MSCs regulate and recruit regulatory T cells (Tregs) in a co-culture approach [24-26]. Tregs themselves have been identified as key players in numerous diseases, among them rheumatoid arthritis [2, 27]; however, until recently they have not been associated with OA pathogenesis [28, 29]. SPTLC1 Although an important number of Phase I/II

studies using MSCs in OA have been started (overview on [30]) and these cells have already been used in small patient series [31], the underlying processes of both the regenerative properties and, more importantly, the immunosuppressive capacities of MSCs in OA, are only poorly understood. The aim of this study, therefore, was to analyse the effect of human MSCs from OA patients on Tregs in an allogeneic lymphocyte co-culture model. We compared MSCs derived from the bone marrow of a joint-adjacent bone and from the synovium of the affected joint to investigate whether the synovial MSCs located within the tissue affected by inflammation exerted different immunomodulatory properties. MSCs were isolated from bone marrow and synovial membrane of 34 patients (age 68 ± 12 years, 19 female and 15 male) that had been collected during total hip arthroplasty for primary OA Kellgren grades III and IV.

Treatment of anaemia in people requiring dialysis who have heart failure should follow the

KHA-CARI Guideline ‘Biochemical and Haematological Targets: Haemoglobin’[1] without modification because of the presence of heart failure (ungraded). Chronic kidney disease and chronic heart failure (CHF) frequently coexist. The mechanisms for this,[2] and a potential classification Protein Tyrosine Kinase inhibitor of this ‘cardiorenal syndrome’,[3] have been reviewed in depth by others. Risk factors such as hypertension and diabetes are common to both CKD and CHF. Many current treatment recommendations for the management of CHF are based on the highest levels of evidence. However, most guidelines make no recommendations specific to patients with CKD. This guideline seeks to fill this gap. Chronic kidney disease is defined as a glomerular filtration rate (GFR) less than 60 mL/min, unless otherwise stated. This is ‘moderate’ Palbociclib purchase (Stage 3 or worse) CKD according to the National Kidney Foundation Kidney Disease Outcomes Quality Initiative (NKF KDOQI) Clinical Practice

Guidelines for Chronic Kidney Disease.[4] However, not all studies providing evidence for this guideline meet the NKF KDOQI criteria of having two measures of kidney function at least 3 months apart. The following definition of CHF stated in the National Heart Foundation (NHF) of Australia Guideline[5, 6] is used for this Guideline: A complex clinical syndrome with typical symptoms (eg, dyspnoea, fatigue) that can occur at rest or on effort that is characterised by objective evidence of an underlying

structural abnormality OR cardiac dysfunction that impairs the ability of the ventricle to fill with or eject blood (particularly during exercise). This guideline does not consider ‘heart failure with reduced ejection fraction’ and ‘heart failure with preserved ejection fraction’ 4-Aminobutyrate aminotransferase separately. The prevalence of CHF or reduced systolic function is increased in patients with CKD compared with people with normal kidney function. In the Chronic Renal Insufficiency Cohort, a history of CHF was reported by 15% of participants with a GFR < 30 mL/min, compared with 5% in participants with GFR > 60 mL/min.[7] Likewise, the prevalence of CKD is very high in CHF patients. In many trial cohorts, this prevalence is over one-third and patients with CHF who also have CKD have a greater mortality risk than patients with CHF and normal kidney function.[8-11] In fact, reduced creatinine clearance was a stronger predictor of adverse outcome than reduced left ventricular ejection fraction (LVEF) in one study.[12] Heart failure is also a significant comorbidity in end-stage kidney disease (ESKD).

The mean age was 42.9 years at time of transplant. For seven patients, the allograft thrombosis was their first kidney transplant and seven of the nine cases had a deceased donor transplant. The initial transplants functioned for selleckchem a mean of 1.67 days and the patients received a second allograft at a mean of 3.1 days after graft failure. All of the re-transplants worked immediately. Four allografts failed after a mean of 52.5 months (2–155 months). Two of these died with

a functioning allograft, one failed owing to chronic allograft nephropathy and one owing to persistent acute cellular rejection. The remaining five patients still have a functioning allograft after a mean of 101.8 months (7–187 months). One year allograft and patient survival after re-transplantation were 87.5% and 100% respectively (after 5 years, both were 57%). Immediate re-transplantation following early kidney transplant thrombosis LY294002 can be a success. It may be considered in selected cases after allograft thrombosis. “
“Apolipoprotein A-I amyloidosis is a rare, autosomal dominant disorder characterized by progressive accumulation of amyloid fibrils in tissues, leading to renal and hepatic disease. We describe the clinical manifestations and pathologic features of kidney disease in three Irish families. This observational

study examines all known cases of chronic kidney disease due to hereditary apolipoprotein A-I amyloidosis in Ireland. Patients were identified by physician interview. In all of the affected individuals the disease was caused by the Gly26Arg heterozygous mutation. Immunohistochemistry confirmed that amyloid deposits were composed of apolipoprotein A-I fibrils. Family trees and clinical data were obtained via analysis of patient

medical records. The vast majority of affected cases had demonstrable kidney disease, with variable liver disease. Renal disease ID-8 most commonly manifested as slowly progressive renal impairment with mild proteinuria. In one kindred, a severe, debilitating peripheral neuropathy was common among affected family members. Histology demonstrated tubulointerstitial fibrosis with amyloid deposition in the medulla. There was very high penetrance within affected families. Of five patients who were transplanted, one transplant was lost after 5 years due to recurrent disease. One patient died from sepsis shortly after transplant. Hereditary apolipoprotein A-I amyloidosis is characterized by slowly progressive renal disease. Amyloid is deposited in the renal medulla highlighting the need to examine the medulla on renal biopsy. Overall, kidney transplantation conferred a survival advantage. “
“We recommend that in patients with chronic kidney disease (CKD), end-stage renal failure (ESRF) and after kidney transplantation, that guidelines for revascularization of the general population be adhered to (1D).

2B–E). The structural integrity of VLPs is required for killing and cytokine secretion functions, since heating disruption of VLPs (95°C condition) decreased both activities (Fig. 2C and E). Internalization of HPV–VLPs in DCs has been shown to induce their activation 22, but it is unknown whether the virus could also specifically enter

into NK cells. Therefore, we investigated VLP uptake by NK cells using CFSE-labeled VLPs. CFSE is a marker that becomes fluorescent only after the removal of the acetate groups by cellular serine esterase, i.e. inside the cells Ruxolitinib molecular weight 23. We performed a kinetic study with CFSE–VLPs at 37°C on NK, CasKi cells and DCs. We observed a weaker fluorescence in NK cells compared with CasKi, although the fluorescence in PF-02341066 chemical structure NK cells reached a plateau very quickly (after 10 min) (Fig. 3A). We also compared the entry into DCs and NK cells derived from the same donor. After 10 min of incubation, we observed a higher fluorescence in NK cells compared with DCs (Fig. 3B). This uptake was not restricted to VLPs from

HPV16, since the VLP entry into NK cells was similar with HPV31– and HPV16–VLPs (Supporting Information Fig. 2A). The VLP entry seemed to require an active process because entry did not occur at 4°C (Supporting Information Fig. 2B). When the VLP structure was disrupted by heating at 95°C, the resulting fluorescence intensity in NK cells was significantly decreased, suggesting that the conformation of VLPs is important for the process of internalization (Supporting Information Fig. 2B). In order to visualize the entry of VLPs into NK cells, we performed confocal and electron microscopy analyses (Fig. 4). oxyclozanide We detected fluorescent VLPs in few large fluorescence spots inside NK cells after 10 min of VLP incubation at 37°C (Fig. 4A) but not in DCs or in CasKi cells (data not shown). After 5 h of incubation, VLPs were observed as being dispersed in the cytoplasm of DCs (Fig. 4B) and of CasKi cells (Fig. 4C). This VLP distribution was not observed in NK cells; even after 10 h of incubation, VLPs were still contained in few large vesicles. Electron microscopy

experiments were performed on NK cells incubated with VLPs (Fig. 4D–F). VLPs were present in large vacuoles (mean diameter: 0.24±0.14 μm, n=22) after 10 min of incubation (Fig. 4D). Similar observations were made after 6 to 18 h and fusion of these vacuoles with the nucleus was not observed (data not shown). At a longer incubation period (18 h), we noticed very large vacuoles, which could come from fusion of smaller vesicles and where VLPs seemed partially degraded (Fig. 4E). We did not observe clathrin-coated vesicles containing VLPs in NK cells as observed in DCs (Fig. 4F) where the vacuole size was smaller (mean diameter: 0.12±0.3 μm, n=6). The membrane ruffles observed by electron microscopy of NK cells in the presence of VLPs (Fig.