Immunogenicity and Protective Capacity of CpG ODN Adjuvanted Alum Adsorbed Bivalent Meningococcal Outer Membrane Vesicle Vaccine.

Invasive meningococcal disease (IMD) is caused by Neisseria meningitidis, with the main serogroups responsible for the disease being A, B, C, W, X, and Y. To date, several vaccines targeting N.meningitidis have been developed albeit with a short-lived protection. Given that MenW and MenB are the most common causes of IMD in Europe, Turkey, and Middle East, we aimed to develop an outer membrane vesicle (OMV) based bivalent vaccine as the heterologous antigen source. Herein, we compared the immunogenicity, and breadth of serum bactericidal assays (SBA) based protective coverage of OMV vaccine to X serotype with existing commercial meningococcal conjugate and polysaccharide (PS) vaccines in a murine model. BALB/c mice were immunized with preclinical batches of the W+B OMV vaccine, either adjuvanted with Alum, CpG ODN or their combinations and compared with a MenACYW conjugate vaccine (NimenrixTM, Pfizer) and a MenB OMV-based vaccine (Bexsero®, GSK), The immune responses were assessed through ELISA and SBA. Antibody responses and SBA titers were significantly higher in the W+B OMV vaccine when adjuvanted with Alum or CpG ODN, as compared to the control groups. Moreover, the SBA titers were not only significantly higher than those achieved with available conjugated ACYW vaccines but also on par with the 4CMenB vaccines. In conclusion, the W+B OMV vaccine demonstrated the capacity to elicit robust antibody responses, surpassing or matching the levels induced by licensed meningococcal vaccines. Consequently, the W+B OMV vaccine could potentially serve as a viable alternative or supplement to existing meningococcal vaccines.

Process development for an effective COVID-19 vaccine candidate harboring recombinant SARS-CoV-2 delta plus receptor binding domain produced by Pichia pastoris.

Recombinant protein-based SARS-CoV-2 vaccines are needed to fill the vaccine equity gap. Because protein-subunit based vaccines are easier and cheaper to produce and do not require special storage/transportation conditions, they are suitable for low-/middle-income countries. Here, we report our vaccine development studies with the receptor binding domain of the SARS-CoV-2 Delta Plus strain (RBD-DP) which caused increased hospitalizations compared to other variants. First, we expressed RBD-DP in the Pichia pastoris yeast system and upscaled it to a 5-L fermenter for production. After three-step purification, we obtained RBD-DP with > 95% purity from a protein yield of > 1 g/L of supernatant. Several biophysical and biochemical characterizations were performed to confirm its identity, stability, and functionality. Then, it was formulated in different contents with Alum and CpG for mice immunization. After three doses of immunization, IgG titers from sera reached to > 106 and most importantly it showed high T-cell responses which are required for an effective vaccine to prevent severe COVID-19 disease. A live neutralization test was performed with both the Wuhan strain (B.1.1.7) and Delta strain (B.1.617.2) and it showed high neutralization antibody content for both strains. A challenge study with SARS-CoV-2 infected K18-hACE2 transgenic mice showed good immunoprotective activity with no viruses in the lungs and no lung inflammation for all immunized mice.

The effect of exosomes released from apheresis platelet concentrates under the impact of gamma irradiation and storage time upon platelet aggregation and hemostasis.

BACKGROUND: Blood components should be gamma-irradiated (γ-IR) in order to prevent transfusion-associated graft-versus-host disease. The aim of this study is to determine the effect of γ-IR and storage time on the exosomes released from apheresis platelet concentrates (aPC) and to investigate their impact on the maximum platelet aggregation (MPA) and hemostasis. MATERIALS AND METHODS: Eight units of aPC were included in this study. These were divided into four equal portions. Two portions were irradiated before storage while the other two were not. Thus, irradiated and non-irradiated aPC samples for storage Days 0 (D0) and 5 (D5) were obtained. Exosomes were isolated from these samples using a commercial kit and were evaluated to ascertain their parent cells by flow cytometry. For the following steps, exosomes were pooled according to their features. Pooled exosomes were then used for aggregometry and thromboelastography. RESULTS: Platelet-derived exosome (PD-EX) levels decreased in D5 compared to D0 in NI-aPC, whereas granulocyte-derived exosome (GD-EX) levels increased. Exosome pools had no effect on MPA compared to saline groups. Exosome pools decreased the time to initial fibrin formation (R), whereas they increased the rate of clot formation (α-angle) and coagulation index (CI) compared to saline groups. DISCUSSION: Storage time and γ-IR each have almost the opposite effects on PD-EX and GD-EX. Exosomes have no impact on MPA, but enhance the clot strength. The impact of exosomes on aPC quality and effectiveness can be ignored or considered as a positive effect.

Leishmania kinetoplast DNA contributes to parasite burden in infected macrophages: Critical role of the cGAS-STING-TBK1 signaling pathway in macrophage parasitemia.

Leishmania parasites harbor a unique network of circular DNA known as kinetoplast DNA (kDNA). The role of kDNA in leishmania infections is poorly understood. Herein, we show that kDNA delivery to the cytosol of Leishmania major infected THP-1 macrophages provoked increased parasite loads when compared to untreated cells, hinting at the involvement of cytosolic DNA sensors in facilitating parasite evasion from the immune system. Parasite proliferation was significantly hindered in cGAS- STING- and TBK-1 knockout THP-1 macrophages when compared to wild type cells. Nanostring nCounter gene expression analysis on L. major infected wild type versus knockout cells revealed that some of the most upregulated genes including, Granulysin (GNLY), Chitotriosidase-1 (CHIT1), Sialomucin core protein 24 (CD164), SLAM Family Member 7 (SLAMF7), insulin-like growth factor receptor 2 (IGF2R) and apolipoprotein E (APOE) were identical in infected cGAS and TBK1 knockout cells, implying their involvement in parasite control. Amlexanox treatment (a TBK1 inhibitor) of L. major infected wild type cells inhibited both the percentage and the parasite load of infected THP-1 cells and delayed footpad swelling in parasite infected mice. Collectively, these results suggest that leishmania parasites might hijack the cGAS-STING-TBK1 signaling pathway to their own advantage and the TBK1 inhibitor amlexanox could be of interest as a candidate drug in treatment of cutaneous leishmaniasis.

Development and preclinical evaluation of virus-like particle vaccine against COVID-19 infection.

BACKGROUND: Vaccines that incorporate multiple SARS-CoV-2 antigens can further broaden the breadth of virus-specific cellular and humoral immunity. This study describes the development and immunogenicity of SARS-CoV-2 VLP vaccine that incorporates the four structural proteins of SARS-CoV-2. METHODS: VLPs were generated in transiently transfected HEK293 cells, purified by multimodal chromatography, and characterized by tunable-resistive pulse sensing, AFM, SEM, and TEM. Immunoblotting studies verified the protein identities of VLPs. Cellular and humoral immune responses of immunized animals demonstrated the immune potency of the formulated VLP vaccine. RESULTS: Transiently transfected HEK293 cells reproducibly generated vesicular VLPs that were similar in size to and expressing all four structural proteins of SARS-CoV-2. Alum adsorbed, K3-CpG ODN-adjuvanted VLPs elicited high titer anti-S, anti-RBD, anti-N IgG, triggered multifunctional Th1-biased T-cell responses, reduced virus load, and prevented lung pathology upon live virus challenge in vaccinated animals. CONCLUSION: These data suggest that VLPs expressing all four structural protein antigens of SARS-CoV-2 are immunogenic and can protect animals from developing COVID-19 infection following vaccination.

Circulating extracellular vesicles of patients with steroid-sensitive nephrotic syndrome have higher RAC1 and induce recapitulation of nephrotic syndrome phenotype in podocytes.

Since previous research suggests a role of a circulating factor in the pathogenesis of steroid-sensitive nephrotic syndrome (NS), we speculated that circulating plasma extracellular vesicles (EVs) are a candidate source of such a soluble mediator. Here, we aimed to characterize and try to delineate the effects of these EVs in vitro. Plasma EVs from 20 children with steroid-sensitive NS in relapse and remission, 10 healthy controls, and 6 disease controls were obtained by serial ultracentrifugation. Characterization of these EVs was performed by electron microscopy, flow cytometry, and Western blot analysis. Major proteins from plasma EVs were identified via mass spectrometry. Gene Ontology classification analysis and Ingenuity Pathway Analysis were performed on selectively expressed EV proteins during relapse. Immortalized human podocyte culture was used to detect the effects of EVs on podocytes. The protein content and particle number of plasma EVs were significantly increased during NS relapse. Relapse NS EVs selectively expressed proteins that involved actin cytoskeleton rearrangement. Among these, the level of RAC-GTP was significantly increased in relapse EVs compared with remission and disease control EVs. Relapse EVs were efficiently internalized by podocytes and induced significantly enhanced motility and albumin permeability. Moreover, relapse EVs induced significantly higher levels of RAC-GTP and phospho-p38 and decreased the levels of synaptopodin in podocytes. Circulating relapse EVs are biologically active molecules that carry active RAC1 as cargo and induce recapitulation of the NS phenotype in podocytes in vitro.NEW & NOTEWORTHY Up to now, the role of extracellular vesicles (EVs) in the pathogenesis of steroid-sensitive nephrotic syndrome (NS) has not been studied. Here, we found that relapse NS EVs contain significantly increased active RAC1, induce enhanced podocyte motility, and increase expression of RAC-GTP and phospho-p38 expression in vitro. These results suggest that plasma EVs are biologically active molecules in the pathogenesis of NS.

TLR ligand loaded exosome mediated immunotherapy of established mammary Tumor in mice.

Tumor-derived exosomes (TEXs) could be harnessed as an immunotherapeutic cancer vaccine. These nanovesicles are inherently possesses rich tumor antigen reservoirs. Due to their undesirable features such as poor or limited immunogenicity as well as facilitation of cancer development via mediating communication between tumor cells TEXs could be transformed into an effective immune adjuvant delivery system that initiates a strong humoral and cell-mediated tumor-specific immune response. Engineering TEXs to harbor immunostimulatory molecules still remains a challenge. Previously, we demonstrated that nucleic acid ligand encapsulated liposomes could trigger synergistic strong humoral, and cell mediated immune responses and provokes tumor regression to that of their standalone counterparts. In this study, we evaluated to immunogenicity of 4T1/Her2 cell-derived exosomes upon loading them with two potent immuno adjuvant, a TLR9 ligand, K-type CpG ODN and a TLR3 ligand, p(I:C). Engineered TEXs co-encapsulating both ligands displayed boosted immunostimulatory properties by activating antigen-specific primary and memory T cell responses. Furthermore, our exosome-based vaccine candidate elicited robust Th1-biased immunity as evidenced by elevated secretion of IgG2a and IFNγ. In a therapeutic cancer model, administration of4T1 tumor derived exosomes loaded with CpG ODN and p(I:C) to animals regress tumor growth in 4T1 tumor-bearing mice. Taken together this work implicated that an exosome-based therapeutic vaccine promoted strong cellular and humoral anti-tumor immunity that is sufficient to reverse established tumors. This approach offers a personalized tumor therapy strategy that could be implemented in the clinic.

Clinical Efficacy of Hormonal and Nonhormonal Agents in the Treatment of Vulvovaginal Atrophy.

OBJECTIVES: Symptomatic local treatment of vaginal atrophy (VA) in menopausal women includes hormonal and nonhormonal preparations. Some women may be reluctant to use vaginal estradiol preparations because of the concern for developing breast cancer and endometrial hyperplasia. Therefore, it is necessary to compare the therapeutic effectiveness of alternative vaginal drugs, such as promestriene, an estrogen agonist, and sodium hyaluronate (NaH), a nonhormonal, water-based agent. METHODS: Ninety-one postmenopausal women diagnosed with symptomatic VA were divided into three groups and treated for 12 weeks; 30 women with vaginal estradiol (VE), 30 women with promestriene, and 31 women with NaH. Composite scoring, vaginal maturation index (VMI), pH, frequency of sexual activity, serum hormone levels, and endometrial thickness were evaluated VA before and after treatment. RESULTS: In the comparison of VA examination findings composite scoring, VMI, and vaginal pH values, three different drugs were found to be effective in the treatment (P < 0.05). The VMI following VE treatment was significantly higher than that after NaH treatment (P = 0.031), whereas the promestriene group had a more positive change than the others in terms of increase in after treatment composite scoring and sexual activity frequency (P = 0.031, P = 0.020). There were no differences between the groups in terms of pre and after treatment serum E2 levels and endometrial thickness. CONCLUSIONS: Based on these findings, we can conclude that the use of promestriene or NaH can prove to be as effective and well tolerated as vaginal estradiol in the symptomatic treatment of vaginal atrophy.

Circulating LL37 targets plasma extracellular vesicles to immune cells and intensifies Behçet's disease severity.

Behçet's disease (BD) activity is characterised by sustained, over-exuberant immune activation, yet the underlying mechanisms leading to active BD state are poorly defined. Herein, we show that the human cathelicidin derived antimicrobial peptide LL37 associates with and directs plasma extracellular vesicles (EV) to immune cells, thereby leading to enhanced immune activation aggravating BD pathology. Notably, disease activity was correlated with elevated levels of circulating LL37 and EV plasma concentration. Stimulation of healthy PBMC with active BD patient EVs induced heightened IL1ß, IFNα, IL6 and IP10 secretion compared to healthy and inactive BD EVs. Remarkably, when mixed with LL37, healthy plasma-EVs triggered a robust immune activation replicating the pathology inducing properties of BD EVs. The findings of this study could be of clinical interest in the management of BD, implicating LL37/EV association as one of the major contributors of BD pathogenesis. Abbreviations: BD: Behçet's disease; EV: extracellular vesicle; BB: binding buffer; AnV: annexin V; autologEV: autologous extracellular vesicles; alloEV: allogeneic extracellular vesicles.

Viral Pneumonias in Forensic Autopsies: Evaluation and Classification of Histopathologic Changes With Microbiologic Correlation.

Lower respiratory infections are commonly due to viruses and are the third largest cause of death. Respiratory tract viruses have a tendency to target the specific regions in the lung and can harm the host via direct effect of the virus and the host's inflammatory response. In this study, relationships between morphologic changes in the lung and the viral agent type isolated in the lung by the polymerase chain reaction technique were investigated. This study was performed retrospectively at 113 autopsy cases in the Council of Forensic Medicine in Istanbul. Slides from the lung tissues diagnosed as interstitial pneumonia and detected viral agent in polymerase chain reaction were evaluated and reviewed under light microscope by 2 pathologists simultaneously according to predetermined bronchiolar, alveolar, and interstitial findings. Alveolar findings were detected in 108 cases (95.6%), whereas interstitial and bronchiolar findings were detected in 91 (80.5%) and 38 (33.6%) cases, respectively. Intra-alveolar edema was the most common alveolar finding. Some findings such as multinucleated syncytial cells and smudge cells can aid the search for etiologic agent. Interstitial inflammation was the most common histopathologic finding in the lung in viral infections and the most prominent clue to viral infections in the lung histopathologically without discrimination of viral agent type.

The effect of erythropoietin on biomechanical properties of the Achilles tendon during the healing process: an experimental study.

BACKGROUND: The aim of this study was to examine the potential biomechanical and histological benefits of systemic erythropoietin administration during the healing of Achilles tendon injury in a rat experimental model. METHODS: Eighty Sprague-Dawley female rats were included in this study. Animals were randomly assigned into two groups with 40 animals in each: erythropoietin group and control group. Then each group was further divided into four subgroups corresponding to four time points with 10 animals in each. A full-thickness cut was made on the Achilles tendon of each animal and then the tendon was sutured with modified Kessler method. Erythropoietin groups received intraperitoneal erythropoietin (500 IU/kg/day) every day at same time throughout the study period, and the control groups received saline in a similar manner. Animals were sacrificed at four time points, and tensile test was performed on each tendon sample to assess maximum load for each sample. In addition, histopathological examination and scoring was done. RESULTS: Both groups had improvement on tensile test (maximum load) over time. However, groups did not differ with regard to maximum load in any of the time points. Similarly, groups did not differ with regard to any of the histopathological scores over time. CONCLUSIONS: The findings of this study do not support the benefit of systemic erythropoietin administration in Achilles tendon healing process. Further evidence from larger experimental studies is required to justify any such potential benefit.

Postmortem diagnosis of cytomegalovirus and accompanying other infection agents by real-time PCR in cases of sudden unexpected death in infancy (SUDI).

As an opportunistic pathogen with high mortality rates, Cytomegalovirus (CMV) may lead to fatal disseminated CMV infection of the premature and newborn; thus necessitating the demonstration of CMV-DNA with clinical history and/or histopathological findings of CMV infection and defining other bacterial and viral infection agents with real-time polymerase chain reaction (RT-PCR) in udden unexpected death in infancy (SUDI) cases as we aimed in this study. 314 (144 female, 170 male) SUDI cases were prospectively investigated from January 2013 to January 2015 in Istanbul Forensic Medicine Institution. The study includes 87 tissue samples of 39 cases for post-mortem histopathological examination of interstitial pneumonia, myocarditis, meningitis, encephalitis, hepatitis, colitis or tubulointerstitial nephritis and/or accompanying chronic sialadenitis. CMV-DNA was found positive in 35 (40.2%) salivary gland, 19 (21.8%) lung, 1 (1.1%) tonsil, and 1 (1.1%) brain tissues. CMV sialadenitis and/or CMV pneumonia associated with other viral and/or bacterial agents were detected in 23 (60%) of 39 infant cases. The demonstration of CMV-DNA would significantly clarify the cause of death and collection of epidemiological data in SUDI cases with clinical history and histopathological findings of CMV infection accompanying chronic CMV sialadenitis. Furthermore, CMV suppresses the immune system, and may predispose to other bacterial and/or viral infections in these cases. Post-mortem molecular investigations are useful in explaining cause of death in SUDI with a suspicion of infection in forensic autopsies.

Hydatid Disease Involved in the Heart, Liver, and Kidney That Caused Sudden Death: Case Report.

Hydatid disease is a parasitic infestation caused by ingestion of eggs of echinococcal species. For Echinococcus granulosus, the definitive host is the dog, and sheeps are the usual intermediate hosts. Humans are accidental intermediate hosts, infected by ingestion of food contaminated with eggs shed by dogs or foxes. The most common organs that hydatid disease encountered are the liver and lungs. Involvement of the kidney is rare and usually accompanies the other organ involvements. Cardiac involvement of echinococcosis is also very rare. We report the case of a 31-year-old woman with a 6-year history of asthma who collapsed after strenuous activity and died despite the interventions carried out. At autopsy, cystic masses were detected in the apex of the heart, in the right kidney, and in the liver. There were no macroscopic pathologic findings in the other organs. Microscopic examination revealed the diagnosis of hydatid cyst in the heart, right kidney, and liver besides medial hypertrophy of the lung vessels. Cause of death was attributed to hydatid cyst and its complications. Patients who have symptoms akin to asthma at clinical presentation have to be further investigated for organic cardiac and pulmonary diseases such as hydatid cyst, especially in endemic countries.

Antienflamatuar and antiadhesive effect of clioquinol.

BACKGROUNDS/AIMS: The aim of this study is to investigate the expected adhesion-modifying effect of clinoquinol which has metal-chelating feature that limits the inflammation and fibroblastic activity. METHODS: A total of 40 Wistar Albino rats were included, which were divided into 5 groups. Group-1 constituted the sham group. Other groups, adhesions were induced by performing cecal abrasion on the rats. For treatment, saline solution was added to Group-2, carboxymethylcellulose was added to Group-3, methylene blue was added to Group-4, and clioquinol was added to Group-5. Blood samples were obtained from the heart to measure IL-10 and TNF α-levels. Adhesions were evaluated both macroscopically and histopathologically. RESULTS: Clioquinol reduced adhesions at significant level via decreasing the inflammation and fibroblastic activity in the adhesion-induced rats. At macroscopic level, carboxymethylcellulose and clioquinol were the most potent agents in reducing adhesions. Nonetheless, significant foreign body-reaction was observed in the carboxymethylcellulose-treated group. CONCLUSIONS: Clioquinol could reduce the formation of intra-abdominal adhesions. It exerts this activity by limiting the inflammation and fibroblastic activity between the intestines and serous surfaces. Furthermore, it does not induce a foreign body reaction. Due to these properties, we conclude that clioquinol can be used as an alternative agent to prevent adhesions.

[Comparison of two different real-time PCR systems in postmortem diagnosis of tuberculosis in paraffin-embedded tissues]. / Postmortem tüberküloz tanisinda parafi ne gömülü dokularda iki farkli gerçek zamanli PCR sisteminin karsilastirilmasi

Tuberculosis (TB) is one of those infections with high morbidity and mortality in all around the world. Hundreds of people died from this disease without diagnosed or due to resistant strains in Turkey. Therefore, it is important to identify postmortem cases who have died from tuberculosis. Molecular methods have been widely used as well as conventional methods in the diagnosis of tuberculosis. The aim of this study was to compare the two different real-time polymerase chain reaction (Rt-PCR) system in the postmortem diagnosis of Mycobacterium tuberculosis infections in paraffin-embedded tissues. A total of 40 paraffin-embedded tissue samples [lung (n= 35), brain (n= 2), heart (n= 2), lymph node (n= 1)] in which histopathologic findings consistent with TB (necrotizing granulomatous inflammation, gelatinous caseous pneumonia, necrotic fibrous nodul) obtained from 37 autopsy cases (31 male, 6 female; age range: 25-85 yrs) were included in the study. Paraffin-embedded tissues were deparafinized with xylene and ethyl alcohol and then DNA isolation was done with QIAsymphony DSP Virus/Pathogen Midi kit in the QIAsymphony device. DNA amplification process was performed by Rt-PCR using the kit Artus® M. tuberculosis RG-PCR in the Rotor-Gene® Q device (Qiagen, Germany). Likewise, after deparafinization process, samples placed in the cartridge and isolation and Rt-PCR was performed by Xpert® MTB/RIF (Cepheid, USA) system, simultaneosly. Seventeen and 20 out of the 40 paraffin-embedded tissues yielded positive results with Qiagen and Xpert system, respectively. M.tuberculosis DNA was found positive in 13 (32.5%) and negative in 16 (40%) of the samples by both of the systems, exhibiting 72.5% (29/40) of concordance. On the other hand, seven (17.5%) samples that were positive with Xpert system yielded negative result with the Qiagen, while four (10%) samples that were positive with Qiagen yielded negative result with the Xpert system. Of the 20 positive cases detected with Xpert MTB/RIF system, 15 were found rifampicin-susceptible, and three were rifampicin-resistant. In two samples in which M. tuberculosis DNA was low positive, rifampicin resistance could not be detected. The identification of M.tuberculosis infections in postmortem cases will contribute epidemiological data in Turkey. In these cases, effective sampling and diagnosing of M.tuberculosis infections by acid-fast stain and culture methods are crucial. However, in cases without microbiological sampling the detection of M.tuberculosis DNA in paraffin-embedded tissues with PCR, although there are differences between PCR systems has diagnostic value. In conclusion, our data indicated that Xpert MTB/RIF system is more favourable to detect M.tuberculosis DNA in paraffin-embedded tissues, with the advantages of determination of rifampicin resistance, and detection of more positive results within a shorter time.

Detection of staphylococcal exotoxins in antrochoanal polyps and chronic rhinosinusitis with nasal polyps.

OBJECTIVE: Staphylococcal exotoxins have been reported in the pathogenesis of many chronic inflammatory diseases. Recent reports have hypothesized that staphylococcal exotoxins might be related to inflammatory mucosal changes seen in chronic sinusitis with nasal polyps (CRS-NPs). Staphylococcal exotoxins have the capacity to act as superantigens (SAgs), bypassing normal antigen processing and directly stimulating a massive inflammatory response. The objective of this study was to analyze polyp tissue samples from patients with antrochoanal polyps (ACPs) and CRS-NPs for the presence of staphylococcal exotoxins. STUDY DESIGN: Prospective cohort study. SETTING: Tertiary medical center. SUBJECTS AND METHODS: Tissue samples were obtained from 29 patients and 16 controls. Thirteen of 29 patients had ACPs, and 16 had CRS-NPs. Specimens were analyzed for the presence of 5 staphylococcal exotoxins (SEA, SEB, SEC, SED, and toxic shock syndrome toxin-1) using enzyme-linked immunosorbent assay (ELISA). RESULTS: At least 1 toxin was detected in 7 of 13 patients with ACPs and in 13 of 16 patients with CRS-NPs, whereas it was detected in only 4 controls. There were no statistically significant differences between ACP and control groups (P = .063). Our results showed a statistically significant association between toxin detection and patients with CRS-NPs (P = .003). CONCLUSION: Inflammatory triggers in ACP remain a subject of debate, and this study does not support the hypothesis that staphylococcal exotoxins may play a role in ACP ethiopathogenesis. Our research is consistent with the possibility of SAgs as etiological agents in the development of bilateral nasal polyposis.