CrELISA: a fast and robust enzyme-linked immunosorbent assay bypassing the need for purification of recombinant protein

A multitude of antigens has been recently identified by screening of cDNA expression libraries derived from human tumors with autologous sera. Using a phage autoantibody assay and small panels of sera derived from cancer patients or controls it has been shown that some of these antigens display cancer-associated autoantibody responses. The diagnostic and prognostic significance of these potentially cancer-related autoantibodies remains unclear until large-scale assays are developed and serological data are available for hundreds of cancer patients and controls. The major bottleneck for the development of large-scale assays are the cloning, expression and the purification of each of the respective antigens. Due to these limitations and despite the potential clinical relevance large-scale autoantibody tests are established for only a few of these tumor antigens. Here we describe an enzyme-linked immunosorbent assay, Crude lysate ELISA (CrELISA), suitable for antigens identified by expression screening based on crude lysates of antigen-expressing bacteria. This assay permits sensitive and specific autoantibody seroscreening without the need of laborious and time-consuming cloning, expression and purification of recombinant proteins. CrELISA is robust and provides a versatile high throughput procedure for the rapid evaluation of multiple antigens in large-scale serology.     

Axillary lymph node cellular immune response to HER-2/neu peptides in patients with carcinoma of the breast.

HER-2/neu peptides have recently been shown to induce a proliferative response by peripheral CD4(+) T cells in breast cancer patients. To investigate potential differences in the local cellular immune response between breast cancer patients with and without nodal metastases, lymphocytes were isolated from axillary lymph nodes from patients with breast cancer, and proliferative and cytokine responses to HER-2/neu peptides were determined. Freshly isolated lymphocytes from lymph nodes of 7 women undergoing surgery for invasive breast cancer were plated at 20 x 10(5) cells per well in triplicate. Cells were stimulated with HER-2/neu peptides at 50 microg/ml and with control antigens. Incorporation of tritium-labeled thymidine was determined 4 days later. The levels of the cytokines interferon-gamma (IFN-gamma), interleukin-4 (IL-4), and IL-10 were determined at priming and at restimulation with HER-2/neu peptides using a cytokine-specific, double-sandwich, enzyme-linked immunosorbent assay (ELISA). Lymphocytes isolated from the axillary lymph nodes of the patients mounted significant cellular immune response to HER-2/neu peptides, manifested by proliferation and specific cytokine elaboration. Proliferative responses to HER-2/neu peptides were seen in lymphocytes of patients with and without overexpression of HER-2/neu in the primary tumor. In some patients, the proliferative response to HER-2/neu peptides in lymphocytes from lymph nodes with metastases was absent or blunted compared with the response in lymphocytes from lymph nodes without metastases from the same patient (p < 0.05). HER-2/neu peptides induced a predominantly T helper type 1 (Th1) pattern of cytokine response in nodal lymphocytes isolated from breast cancer patients. A Th1-specific cytokine production pattern was maintained at priming and restimulation with HER-2/neu peptides and was amplified with IL-12 costimulation. These results indicate that HER-2/neu peptides can activate T cells in draining lymph nodes from women with invasive breast cancer. This activation is associated with a predominantly Th1 cytokine response, which suggests that conditioning with HER-2/neu peptides may be of value in the development of breast cancer vaccines.     

Chemokine receptor CXCR4 expression in breast cancer as a potential predictive marker of isolated tumor cells in bone marrow

Interactions between the CXCR4 chemokine receptor in breast cancer cells and the ligand CXCL12/SDF-1α are thought to play an important role in breast cancer metastases. In this pilot study, CXCR4 expression along with other biomarkers including HER2-neu and EGFR, were measured in primary tumor samples of patients with operable breast cancer to test whether any of these biomarkers alone and in combination could indicate breast cancer with high likelihood of metastasizing to bone marrow. Cytokeratin (CK) positive cells in bone marrow were identified by flow-cytometry following enrichment with CK 7/8 antibody-coupled magnetic beads. Primary tumors (n = 18) were stained with specific antibodies for CXCR4, HER2-neu, EGFR, and PCNA using an indirect avidin–biotin horseradish peroxidase method. The majority of the patients had T2/T3 tumors (72%), or lymph node involvement (67%) as pathologic characteristics that were more indicative of high-risk breast cancer. High CXCR4 cytoplasmic expression was found in 7 of 18 patients (39%), whereas 6 of 18 patients (33%) were found to have CK positivity in bone marrow. The median number of CK⁺ cells was 236 (range, 20–847) per 5 × 10⁴ enriched BM cells. The presence of CK⁺ cells in bone marrow was found to be associated with increased expression of CXCR4 alone or in addition to EGFR and/or HER2-neu expression (P = 0.013, P = 0.005, and P = 0.025, respectively) in primary tumors. Furthermore, three patients with high CK positivity (>236 CK⁺ per 5 × 10⁴ enriched bone marrow cells) in bone marrow exclusively expressed high levels of CXCR4 with EGFR/HER2-neu (P = 0.001). Our data suggest that high CXCR4 expression in breast cancer may be a potential marker in predicting isolated tumor cells in bone marrow. CXCR4 coexpression with EGFR/HER2-neu might further predict a particular subset of patients with high CK positivity in bone marrow.     

Survivin expression in non-small-cell lung carcinomas: correlation with apoptosis and other apoptosis-related proteins, clinicopathologic prognostic factors and prognosis.

The role of survivin that regulates the biological behavior of non-small-cell lung carcinoma (NSCLC) is still controversial. We aimed to investigate survivin expression in NSCLC and to define any correlation with expressions of p53, bcl-2, bax, apoptotic index (AI), tumor cell proliferation, clinicopathologic variables, and overall survival. Tumors of 63 patients with NSCLC were examined for expressions of survivin, p53, bcl-2, bax, and Ki-67 by immunohistochemistry. AI was also evaluated. Results for each antibody were correlated with each other, and with clinicopathologic variables including age, sex, histologic subtype, TNM (T: primary tumor, N: regional lymph node metastasis, M: distant metastasis) stage, lymph node status, smoking history, and prognosis. Nuclear survivin expression was inversely correlated with p53 expression (P = 0.04, r = - 0.367), and tumor stage (P = 0.03, r = - 0.273), and positively correlated with tumor cell proliferation (P = 0.009, r = 0.329). Cytoplasmic survivin expression positively correlated with smoking history (P = 0.02, r = 0.282). Survivin/bax ratio was inversely correlated with AI (r: - 0.004). By Kaplan-Meier analysis, TNM stage (P < or = 0.001), lymph node metastasis (P = 0.04), and Ki-67 index (P < or = 0.001) were associated with survival, whereas survivin was not. In multivariate analysis, only TNM stage was an independent predictor. Although survivin and other apoptosis-related protein expressions fail to predict the clinical outcome, the present findings suggest that survivin is involved in tumor cell apoptosis and proliferation and may play a role in critical steps of cancer progression in NSCLC.     

Mutagenic potentials of dental cements as detected by the Salmonella/microsome test

The potential mutagenicity of a zinc phosphate (Poscal), a polycarboxylate (Aqualox) and glass ionomer cements with (Argion) and without (Meron) silver reinforcement were characterized by employing the Ames Salmonella/microsome test. The materials were eluted in dimethyl sulphoxide or physiologic saline and the aliquots were used either immediately or after an incubation period of 24h at 37 degrees C. Mutagenic effects of the materials were tested on Salmonella typhimurium strains TA 98, TA 100, TA 102 and TA 1535 using the standard plate incorporation assay, and in the presence or absence of S9 fraction from rat liver. Poscal and Aqualox elicited mutagenic effects on S. typhimurium TA 98 and TA 1535, whereas Meron exhibited mutagenic effects on S. typhimurium TA 98. No mutagenic effects were detected for Argion. The type of solvent, dose of the material and incubation as well as the interactions between these factors exhibited varying degrees of influences on the mutagenic activities of the cements (P<0.05 and P<0.1). We conclude that zinc phosphate, polycarboxylate, and glass ionomer cements may have possible mutagenic activities.     

Information Capacity of the Corticospinal Tract Recordings as a Neural Interface

Recording the motor output of the central nervous system from the cervical spinal cord was investigated as a method of generating voluntary command signals, potentially to be used in quadriplegic individuals. Corticospinal volleys evoked by motor cortex stimulation were recorded from the spinal cord surface with multicontact electrodes in anesthetized cats. The multicontact recordings were analyzed for their information-carrying capacity as a neural interface. Neural signals resulting from the stimulation of various points in the motor cortex were considered as symbols of an alphabet that were sent through a discrete information channel. The information capacity of this channel at the thermal noise level of the electrode contacts was calculated. The maximum information rate was 1.57 bits in a trial for a 4-symbol alphabet. The background noise that reduces the information rate to 50% of its maximum theoretical value was defined as the half-bitrate-noise-tolerance (HBR-NoiseTol) and used as a measure of symbol distinguishability. The HBR-NoiseTol for all trials on average was 24 +/- 12%, 18 +/- 10%, and 15 +/- 9% for interfaces with 2-, 3-, and 4-symbol alphabets (n = 11 trials). The average peak-to-peak amplitude of the neural volleys was 13.5 +/- 6.7 microV (n = 11). These results suggest that the corticospinal signals can be recorded with spatial selectivity from the spinal cord surface and thus warrant further investigation of their potential use for a spinal cord-computer interface.     

Pain during sexual activity and ejaculation following hernia repair: A retrospective comparison of transabdominal preperitoneal versus Lichtenstein repair

Pain during sexual activity and ejaculation are the unspoken long-term complications of groin hernia repair. Laparoscopic surgical techniques are associated with decreased post-operative pain and earlier return to daily activities, but its effect on these complications is unclear. This study aims to investigate the effect of transabdominal preperitoneal repair (TAPP) on de-novo pain during sexual intercourse and ejaculation and to compare with open repair. For this reason, two groups were determined according to the surgical technique: the Lichtenstein repair and the TAPP groups and a questionnaire was sent to the patients a minimum of 6 months following the surgery. A total of 317 patients included, as 115 in TAPP and 202 in Lichtenstein repair group. No significant difference was observed concerning pre-operative pain during sexual activity and ejaculation in both groups (p = .75, p = .56). Following the surgery, the number of patients experiencing painful sexual activity was significantly higher in the Lichtenstein repair group compared to the TAPP group (19.3% vs. 11.3%, respectively, p = .03). The post-operative painful ejaculation rate was also significantly lower for the TAPP group (p = .04). The lower rates of post-operative dysejaculation and pain during sexual activity can be achieved with the advantage of laparoscopic surgery.     

Evaluation of the teratogenic effects of occupational hazards during pregnancy

The Hungarian Case-Control Surveillance System of Congenital Anomalies, 1980-1987 contains 10,937 index patients affected by congenital anomalies and 10,937 matched control cases. Their comparative analysis indicated a significantly higher rate of some congenital anomaly types in the offspring of women worked in agriculture and "other" categories. However, a more detailed analysis of data set (according to different working places) is needed for the improvement of health protection.     

Formate metabolism in young swine

Formate generated from methanol metabolism in vivo is the chemical entity responsible for the development of the methanol toxicity syndrome in the monkey. Compared to rats, monkeys are in a state of folate deficiency. This leads to a decreased ability to dispose of formate generated leading to its accumulation and the subsequent development of the classic symptoms of methanol toxicity. Rats possess a more efficient folate system; therefore, they metabolize formate very readily and do not exhibit methanol toxicity symptoms. In this report, the hepatic folate content and the ability to handle a formate "load" were evaluated in another animal species, the pig. The results obtained indicate that the pig, compared to all other species studied, has extremely low levels of folates and very low levels of a key enzyme in the folate pathway, namely 10-formyl H4folate dehydrogenase. Also the pig's ability to dispose of formate was extremely limited and slower than that observed in rats or monkeys. These results suggest that the pig may be a suitable animal model for studying formate metabolism and possibly methanol toxicity.     

The “vaccine” hubbub: Viral load comparisons of SARS-CoV-2 Delta and Omicron variants against different vaccine–booster vaccine combinations

There is a significant body of evidence showing that efficient vaccination schemes against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is helping control the coronavirus disease 2019 (COVID-19) pandemic. However, this goal cannot be achieved without real world data highlighting the impact of vaccines against viral spread. In this study, we have aimed at differentially investigating the impact of COVID-19 vaccines (CoronaVac, Pfizer/BioNTech, Astra/Zeneca Oxford, Janssen) used in North Cyprus in limiting the viral load of Delta and Omicron variants of SARS-COV-2. We have utilized real-time quantitative polymerase chain reaction cycle threshold values (Ct values) as a proxy of viral load of the two SARS-CoV-2 variants. Our results indicate that the administration of at least two doses of the messenger RNA-based Pfizer/BioNTech vaccine leads to the lowest viral load (highest Ct values) obtained for both Omicron and Delta variants. Interestingly, regardless of the vaccine type used, our study revealed that Delta variant produced significantly higher viral loads (lower Ct values) compared with the Omicron variant, where the latter was more commonly associated with younger patients. Viral spread is a crucial factor that can help determine the future of the pandemic. Thus, prioritizing vaccines that will play a role in not only preventing severe disease but also in limiting viral load and spread may contribute to infection control strategies.