Stent Scraping for Histology: An Alternative Method for Obtaining Tissue to Rule out Neoplasia

Despite improvement in diagnostic modalities, confirmation of a histologic diagnosis of cancer of the biliary tree and pancreas remains elusive. Attempts to collect positive cytology specimens from vigorous brushings or washings obtained at endoscopy or percutaneously are often unsuccessful. In our unit, we have increased the yield by obtaining tissue scraped from prostheses that have been previously placed in either the bile duct or the pancreatic duct. The stents are first flushed with saline to collect cytology specimens, after which, they are bisected and scraped, and these contents are prepared in a manner similar to that used to prepare biopsy samples. Twelve of 16 scraped samples, 9 bile duct and 3 pancreas, were positive for adenocarcinoma. The cytology specimens were positive in only 4 of the 12. We recommend this method of sampling from material contained within prostheses as an adjunct when previous brushings, washings, or biopsies are negative.     

Anti-inflammatory effects of hydroalcoholic extract and two biflavonoids from Garcinia gardneriana leaves in mouse paw oedema

Garcinia gardneriana (Planch. & Triana) Zappi (Clusiaceae) is widely distributed in Brazil and used in folk medicine to treat inflammation, pain, and urinary tract and other infections. However, very few studies have analyzed these therapeutic effects. In this study, the anti-inflammatory effects of the hydroalcoholic extracts from Garcinia gardneriana (HEGG) and some of its isolated biflavonoids were evaluated. The results showed that HEGG from the leaves, bark and seeds reduced carrageenan-induced mouse paw inflammation, in addition to diminishing the myeloperoxidase activity in the stimulated tissues. The reduction of neutrophil infiltration by treatment with the HEGG from leaves was confirmed by histology. The leaf extract also reduced the paw oedema evoked by bradykinin, histamine, prostaglandin E2 and 12-O-tetradecanoylphorbol acetate. However, it partially decreased substance P and compound 48/80-caused paw oedema, without any influence on the arachidonic acid-induced oedema. Both of the isolated compounds, fukugetin and GB-2a, prevented the carrageenan-induced paw oedema. In conclusion, this study showed important anti-inflammatory effects of HEGG through its interaction with different intracellular signaling pathways, without interfering with the formation of arachidonic acid (AA) metabolites. These characteristics, in addition to the wide distribution and culturing ease of the plant, confirm its popular use and highlight its promise in the development of new anti-inflammatory drugs.     

Step‐down approach using either cyclosporin A or methotrexate as maintenance therapy in early rheumatoid arthritis

Objective

To evaluate the feasibility and outcome of the step‐down approach using either cyclosporin A (CSA) or methotrexate (MTX) as maintenance therapy following 6 months treatment with these 2 agents in combination in early, nonerosive rheumatoid arthritis (RA).

Methods

Fifty‐seven patients younger than 65 years with early, nonerosive RA were first treated with CSA and MTX in combination for 6 months. They were then randomly stepped down to single‐agent maintenance treatment for another 18 months. Safety, clinical efficacy, survival on treatment, and radiographic progression were evaluated.

Results

When being treated with combination therapy, 7 of the 57 patients (12.3%) withdrew because of adverse events. Of the remaining 50 patients, 42 (84.0%) were American College of Rheumatology (ACR) 20% responders, 30 (60.0%) were ACR 50% responders, and 23 (46.0%) were ACR 70% responders. At month 6, 22 patients were randomized to CSA and 27 to MTX. During this trial period, the treatment was discontinued by 16 patients taking CSA (mainly because of loss of efficacy) and by 4 taking MTX. At month 24, the probability (± SEM) of survival on treatment was 0.273 ± 0.09 for CSA and 0.852 ± 0.07 for MTX. Of the 6 CSA patients who completed the trial, 4 (66.7%) were ACR 20% responders, and 3 (50%) were both ACR 50% and ACR 70% responders. Of the 23 completers in the MTX arm, 21 (91.3%) were ACR 20% responders, 18 (78.3%) were ACR 50%, and 10 (43.5%) were ACR 70% responders. The treatment was not responsible for severe adverse events. Radiography showed a slow progression in the damage score and number of eroded joints in both treatment groups.

Conclusion

Stepping down to single agent maintenance therapy following 6 months of combination treatment with CSA and MTX in early RA was only successful with MTX. Because this treatment did not prevent some radiographic progression, other approaches (e.g., step‐up approach) may be more appropriate in early RA.

     

Induction of broadly neutralizing antibodies using a secreted form of the hepatitis C virus E1E2 heterodimer as a vaccine candidate

Hepatitis C virus (HCV) is a global disease burden, and a preventive vaccine is needed to control or eradicate the virus. Despite the advent of effective antiviral therapy, this treatment is not accessible to many patients and does not prevent reinfection, making chronic hepatitis C an ongoing global health problem. Thus, development of a prophylactic vaccine will represent a significant step toward global eradication of HCV. HCV exhibits high genetic variability, which leads frequently to immune escape. However, a considerable challenge faced in HCV vaccine development is designing an antigen that elicits broadly neutralizing antibodies. Here, we characterized the immunogenicity of a vaccine based on a soluble, secreted form of the E1E2 envelope heterodimer (sE1E2.LZ). Sera from mice immunized with sE1E2.LZ exhibited an anti-E1E2–specific response comparable to mice immunized with membrane-bound E1E2 (mbE1E2) or a soluble E2 ectodomain (sE2). In competition-inhibition ELISA using antigenic domain-specific neutralizing and nonneutralizing antibodies, sera from sE1E2.LZ-immunized mice showed nearly identical or stronger competition toward neutralizing antibodies when compared with mbE1E2. In contrast, sera from mice immunized with sE2, and to a lesser extent mbE1E2, competed more effectively with nonneutralizing antibodies. An assessment of neutralization activity using both HCV pseudoparticles and cell culture–derived infectious HCV showed that immunization with sE1E2.LZ elicited the broadest neutralization activity of the three antigens, and sE1E2.LZ induced neutralization activity against all genotypes. These results indicate that our native-like soluble glycoprotein design, sE1E2.LZ, induces broadly neutralizing antibodies and serves as a promising vaccine candidate for further development.

Hepatitis C virus (HCV) is a global disease burden, with an estimated 71 million people infected worldwide (1, 2). Roughly 75% of HCV infections become chronic (3–5), and in severe cases can result in cirrhosis or hepatocellular carcinoma (6). Viral infection can be cured at high rates by direct acting antivirals (DAAs), but several issues have blunted their effectiveness in eradicating HCV. In particular, multiple public health and financial barriers (7, 8) restrict access to DAAs in areas with high incidence of infection and DAAs do not prevent reinfection. Moreover, HCV infection is largely asymptomatic and often does not generate sterilizing immunity, thereby contributing to reinfection or continued disease progression (7, 9, 10). Collectively, these issues have resulted in a continued rise in HCV infections.Acute HCV infections can be cleared by host immunity in ∼25% of cases. Among individuals who clear their first infection, the rate of clearance rises to 80% for subsequent infections, indicating an effective immune memory response (11–14). This type of natural protective immunity to HCV requires the induction of broadly neutralizing antibodies to E1E2 ectodomains and T cell responses to the structural and nonstructural proteins (15–17). The above clinical observations suggest that, if a vaccine candidate could induce broadly neutralizing antibody and cell-mediated immune responses equivalent to that seen in spontaneous clearance, such a vaccine would be highly effective at preventing HCV infection. An HCV vaccine therefore remains an essential proactive measure to protect against viral spread, yet vaccine developments against the virus have been unsuccessful to date (17, 18).A number of challenges exist that have thus far limited progress toward developing a prophylactic vaccine against HCV. One major challenge in developing a successful vaccine for HCV has been the remarkable genetic diversity of the virus which has six major genotypes (genotypes 1 to 6), in addition to two less-common genotypes (19) (genotypes 7 and 8), and intragenotypic diversity resulting in 90 total subtypes (20). Moreover, shielding of important neutralizing epitopes with glycans (21, 22), and the presence of immunodominant nonneutralizing epitopes (23–26) deflect the immune response from conserved regions that mediate virus neutralization. Multiple studies in chimpanzees and humans have used E1E2 formulations to induce a humoral immune response, but their success in generating high titers of broadly neutralizing antibody (bnAb) responses has been limited. In particular, immunological assessment in chimpanzees of an E1E2 vaccine produced superior immune responses as compared with E2 administered alone and resulted in sterilizing immunity against homologous virus challenge (27, 28), but with less cross-neutralization capacity against heterologous isolates (29). In addition, an E1E2 formulation tested in humans is well-tolerated (30). However, due to the limited neutralization breadth observed in the human clinical trial (31, 32), using native E1E2 as a vaccine is not likely to provide sufficient protection from HCV infection. Rather, optimization of E1E2 to improve its immunogenicity and capacity to elicit bnAbs through rational design appears to be the preferred path for developing an effective B cell-based vaccine (33).An additional bottleneck contributing to the difficulty in generating protective B cell immune responses required for an effective HCV vaccine is preparation of a homogeneous E1E2 antigen. HCV envelope glycoproteins E1 and E2 form a heterodimer on the surface of the virion (34–36). Furthermore, E1E2 assembly has been proposed to form a trimer of heterodimers (37) mediated by hydrophobic C-terminal transmembrane domains (TMDs) (36, 38, 39) and interactions between E1 and E2 ectodomains (40–42). These glycoproteins are necessary for viral entry and infection, as E2 attaches to the CD81 and scavenger receptor type B class I (SR-B1) coreceptors as part of a multistep entry process on the surface of hepatocytes (43–46). Neutralizing antibody (nAb) responses to HCV infection target epitopes in E1, E2, or the E1E2 heterodimer (25, 47–52). A significant impediment to the uniform production of an immunogenic E1E2 heterodimer that could be utilized for vaccine development is the association of the antigen with the membrane via the TMDs (36, 53). Progress has been made in the production and purification of the membrane-bound E1E2 complex via immunoaffinity purification (54, 55) or the use of tags that allow protein A (56) or anti-Flag (57) chromatography. While these methods produce high-quality samples, they all involve harsh elution conditions. How such conditions might influence sample quality at a scale required for vaccine trials is unclear. Furthermore, intracellular expression and membrane extraction limits the ability to produce large quantities of sufficient homogeneity required for both basic research and vaccine production.In contrast, viral glycoproteins of influenza hemagglutinin (58), respiratory syncytial virus (RSV) (59), SARS-CoV-2 (60), and others (61, 62) have been stabilized in soluble form using a C-terminal attached foldon trimerization domain to facilitate assembly. In addition, HIV gp120-gp41 proteins have been designed as soluble SOSIP trimers in part by introducing a furin cleavage site to facilitate native-like assembly when cleaved by the enzyme (63, 64). Recent efforts have made strides toward liberating the E1E2 complex from the membrane in its native form (65, 66). In particular, our previous work (66) showed that a soluble E1E2 (sE1E2) using the Fos/Jun leucine zipper (LZ) coiled-coil as a scaffold (sE1E2.LZ) is antigenically intact, as the protein is recognized by E1E2-specific mAbs AR4A and AR5A (67). Moreover, sE1E2.LZ elicited nAbs in mice immunized with the antigen, making this scaffold a promising potential platform for engineering of additional HCV vaccine candidates.Here, we describe the immunogenicity of our native-like secreted E1E2 construct sE1E2.LZ and compare it with the membrane-bound E1E2 complex (mbE1E2) and a secreted form of the E2 ectodomain (sE2). Immunization of mice with sE1E2.LZ produced sera possessing anti-E1E2 antibodies at levels comparable to mice immunized with mbE1E2 or sE2. Moreover, the antibody response in sE1E2.LZ-immunized mice is skewed more toward nAbs relative to non-nAbs than the other two antigens. Remarkably, sera from sE1E2.LZ-immunized mice exhibited broader neutralization activity than either mbE1E2 or sE2 when assessed using both pseudotyped HCV particles (HCVpp) and cell culture-derived HCV (HCVcc), suggesting that this sE1E2 platform represents a favorable starting point for developing scaffolded E1E2 vaccine candidates.     

ATM protein and p53-serine 15 phosphorylation in ataxia-telangiectasia (AT) patients and at heterozygotes

ATM (ataxia-telangiectasia mutated) gene plays a central role in the DNA-damage response pathway. We characterized the ATM protein expression in immortalized cells from AT and AT-variant patients, and heterozygotes and correlated it with two ATM-dependent radiation responses, G1 checkpoint arrest and p53-Ser 15 phosphorylation. On Western blots, the full-length ATM protein was detected in eight of 18 AT cases, albeit at 1-32% of the normal levels, whereas a truncated ATM protein was detected in a single case, despite the prevalence among cases of truncation mutations. Of two ataxia without telangiectasia [A-(T)] cases, one expressed 20% and the other approximately 70% of the normal ATM levels. Noteworthy, among ten asymptomatic heterozygous carriers for AT, normal amounts of ATM protein were found in one and reduced by 40-50% in the remaining cases. The radiation-induced phosphorylation of p53 protein at serine 15, largely mediated by ATM kinase, was defective in AT, A(-T) and in 2/4 heterozygous carriers, while the G1 cell cycle checkpoint was disrupted in all AT and A(-T) cases, and in 3/10 AT heterozygotes. Altogether, our study shows that AT and A(-T) cases bearing truncation mutations of the ATM gene can produce modest amounts of full-length (and only rarely truncated) ATM protein. However, this limited expression of ATM protein provides no benefit regarding the ATM-dependent responses related to G1 arrest and p53-ser15 phosphorylation. Our study additionally shows that the majority of AT heterozygotes express almost halved levels of ATM protein, sufficient in most cases to normally regulate the ATM-dependent DNA damage-response pathway.     

Dioxins and Related Compounds in Human Breast Milk Collected Around Open Dumping Sites in Asian Developing Countries: Bovine Milk as a Potential Source

In this study, concentrations of dioxins and related compounds (DRCs)—such as polychlorinated dibenzo-p-dioxins, polychlorinated dibenzofurans, and coplanar polychlorinated biphenyls—were found in human breast milk from women living near dumping sites of municipal waste and reference sites in India, Cambodia, Vietnam, and the Philippines during 1999 to 2000. DRCs were detected in all human breast milk samples analyzed, demonstrating that residents in these Asian developing countries have been exposed to these contaminants. In India, the concentrations of DRCs in human breast milk from women living near the investigated dumping site were notably higher than those from women living near reference sites and from women in other Asian developing countries. Toxic equivalent quantity (TEQ) levels of DRCs were comparable with or higher than those reported in the general populations of developed countries since 1990. In contrast, levels of these contaminants in human breast milk in women from Cambodia and Vietnam were not significantly different between milk from women living near the dumping and reference sites. These results indicate that significant pollution sources for DRCs are present in Indian dumping sites and that residents there have been exposed to relatively higher levels of these contaminants. TEQ levels in human breast milk from the dumping site in India tended to decrease with an increase in the number of previous deliveries by mothers, whereas no significant relationship was observed in Cambodia, Vietnam, or the Philippines. This suggests that mothers who have been exposed to relatively high levels of DRCs transfer greater amounts of these contaminants to the first infant than later ones through breast-feeding, which in turn implies that the first children of these mothers might be at higher risk from DRCs. When the residue levels of DRCs in bovine milk collected from the Indian dumping site and reference sites were examined, TEQ levels in bovine milk from the dumping site were higher than those from reference sites. This result suggests that bovine milk is a potential source of DRCs for residents living near the dumping site in India. To our knowledge, this is the first comprehensive study on exposure to DRCs of residents living in proximity to open dumping sites of municipal waste in Asian developing countries.     

Step-down approach using either cyclosporin A or methotrexate as maintenance therapy in early rheumatoid arthritis

OBJECTIVE: To evaluate the feasibility and outcome of the step-down approach using either cyclosporin A (CSA) or methotrexate (MTX) as maintenance therapy following 6 months treatment with these 2 agents in combination in early, nonerosive rheumatoid arthritis (RA). METHODS: Fifty-seven patients younger than 65 years with early, nonerosive RA were first treated with CSA and MTX in combination for 6 months. They were then randomly stepped down to single-agent maintenance treatment for another 18 months. Safety, clinical efficacy, survival on treatment, and radiographic progression were evaluated. RESULTS: When being treated with combination therapy, 7 of the 57 patients (12.3%) withdrew because of adverse events. Of the remaining 50 patients, 42 (84.0%) were American College of Rheumatology (ACR) 20% responders, 30 (60.0%) were ACR 50% responders, and 23 (46.0%) were ACR 70% responders. At month 6, 22 patients were randomized to CSA and 27 to MTX. During this trial period, the treatment was discontinued by 16 patients taking CSA (mainly because of loss of efficacy) and by 4 taking MTX. At month 24, the probability (+/- SEM) of survival on treatment was 0.273 +/- 0.09 for CSA and 0.852 +/- 0.07 for MTX. Of the 6 CSA patients who completed the trial, 4 (66.7%) were ACR 20% responders, and 3 (50%) were both ACR 50% and ACR 70% responders. Of the 23 completers in the MTX arm, 21 (91.3%) were ACR 20% responders, 18 (78.3%) were ACR 50%, and 10 (43.5%) were ACR 70% responders. The treatment was not responsible for severe adverse events. Radiography showed a slow progression in the damage score and number of eroded joints in both treatment groups. CONCLUSION: Stepping down to single agent maintenance therapy following 6 months of combination treatment with CSA and MTX in early RA was only successful with MTX. Because this treatment did not prevent some radiographic progression, other approaches (e.g., step-up approach) may be more appropriate in early RA.     

Evaluation of von Willebrand factor in COPD patients

OBJECTIVE:

To compare the absolute serum von Willebrand factor (vWF) levels and relative serum vWF activity in patients with clinically stable COPD, smokers without airway obstruction, and healthy never-smokers.

METHODS:

The study included 57 subjects, in three groups: COPD (n = 36); smoker (n = 12); and control (n = 9). During the selection phase, all participants underwent chest X-rays, spirometry, and blood testing. Absolute serum vWF levels and relative serum vWF activity were obtained by turbidimetry and ELISA, respectively. The modified Medical Research Council scale (cut-off score = 2) was used in order to classify COPD patients as symptomatic or mildly symptomatic/asymptomatic.

RESULTS:

Absolute vWF levels were significantly lower in the control group than in the smoker and COPD groups: 989 ± 436 pg/mL vs. 2,220 ± 746 pg/mL (p < 0.001) and 1,865 ± 592 pg/mL (p < 0.01). Relative serum vWF activity was significantly higher in the COPD group than in the smoker group (136.7 ± 46.0% vs. 92.8 ± 34.0%; p < 0.05), as well as being significantly higher in the symptomatic COPD subgroup than in the mildly symptomatic/asymptomatic COPD subgroup (154 ± 48% vs. 119 ± 8%; p < 0.05). In all three groups, there was a negative correlation between FEV₁ (% of predicted) and relative serum vWF activity (r² = −0.13; p = 0.009).

CONCLUSIONS:

Our results suggest that increases in vWF levels and activity contribute to the persistence of systemic inflammation, as well as increasing cardiovascular risk, in COPD patients.