Enzyme-linked Immunofluorescence Assay Research Articles

Differences in Ca 15-3 and Homocysteine Levels in Patients with Benign Tumor and Breast Cancer

More than 25% of women have complaints related to breast lumps during their lifetime and approximately 12% have breast cancer. Cancer antigen 15-3 and homocysteine can be used as biomarkers in breast cancer. Breast cancer produces pro-inflammatory cytokines that can lead to increased CA 15-3 levels and serum homocysteine levels. Objective: To investigate the differences in CA 15-3 and homocysteine levels between patients with benign tumors and breast cancer. Methods: Observational analytic study with a cross-sectional approach in 30 benign breast tumor patients and 31 breast cancer patients. Measurement of CA 15-3 levels using enzyme linked immunofluorescent assay (ELFA) and homocysteine using competitive enzyme linked immunosorbant assay (ELISA). Comparative tests used independent T test and Mann-Whitney test. The study was conducted in 2023 at Kariadi Hospital Semarang and Ken Saras Hospital Ungaran. Results: CA 15-3 levels in the benign breast tumor group had a median of 11.74 (2.00-40.00) U/mL, while the breast cancer group was 18.66 (7.22-168.97) U/mL (p = 0.001). Homocysteine levels in benign breast tumors had a median of 3.05 (0.10-11.50) µmol/L and in the breast cancer group was 9.90 (3.70-17.20) µmol/L (p = 0.0001). Conclusion: CA 15-3 and homocysteine levels were lower in benign breast tumor patients compared with breast cancer patients.

Tumor cell density dependent IL-8 secretion induces the fluctuation of tregs/CD8 + T cells infiltration in hepatocellular carcinoma: one prompt for the existence of density checkpoint

BackgroundTumor cell density is a basic pathological feature of solid tumors. Chemotherapy, radiotherapy, and targeted therapy reduce tumor cell density, whereas unrestricted tumor cell proliferation promotes this feature. The impact of tumor cells on the microenvironment following changes in tumor cell density is still unclear. In this study, we focused on the response of key immune cell subsets to tumor cell density in hepatocellular carcinoma (HCC).MethodsWe determined the density of tumor and immune cells in the same area by section staining. We then identified potential mediators using polymerase chain reaction (PCR), enzyme-linked immunofluorescence assay (ELISA), 3D and co-culture, flow cytometry, and lentivirus intervention. The mechanism of lactate promotion was verified using lactate tests, bioinformatics, western blotting, and the above methods. The IL-8/DAPK1/lactate/regulatory T cell (Treg) axis was verified using a mouse liver cancer model. Tumor mutation burden was calculated using maftools in R.ResultsWe found that the Treg/CD8 + T cell ratio is not consistent with tumor cell density in HCC, and a decreased Treg/CD8 + T cell ratio in the range of 5000–6000 cells/mm2 may elicit the possibility for immunotherapy in an immunosuppressive microenvironment. We showed that IL-8 mediates this immune fluctuation and promotes the infiltration of Tregs through the DAPK1/pyruvate kinase activity/lactate axis in HCC. Based on tumor ploidy and mutation burden data, we discussed the potential significance of immune fluctuation in the homeostasis of HCC mutation burden and proposed a “density checkpoint” and “entropy model” to describe this phenomenon.ConclusionsIn summary, we report the mode of infiltration of Tregs/CD8 + T cells in response to tumor cell density and provide a new theoretical basis for IL-8 as a therapeutic target and the selection of an immunotherapy window in HCC.

Prevalence of SARS -CoV-2 IgG/IgM antibodies among patients in Zakho City, Kurdistan, Iraq.

In 2019, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the agent of Coronavirus Disease 2019 (COVID-19), spread all over the world. This global pandemic spread rapidly to more than 195 countries and caused over 200 million infections with a mortality rate of 2%. This study aimed to detect seropositivity against the SARS-CoV-2 virus among outpatients, symptomatic and asymptomatic individuals. A total of 489 individuals of age 5-70 years (mean 38.0 ± 17 SD) were enrolled for a cross-sectional study. They were tested for presence of SARS-CoV-2 IgM/IgG antibodies in serum samples using Enzyme-linked Immunofluorescent Assay (ALFA). A higher seroprevalence was recorded for IgM among females (34.96%) than males (28.83%). In the case of IgG, and IgG + IgM, both males and females had similar values. A significant correlation was identified between seropositivity and age; higher seropositivity (IgG, IgM, and IgG + IgM) was recorded in age groups 51-60 and ≥ 61 years, relative to the younger age groups. No significant correlation was found between symptomatic and asymptomatic individuals. No significant correlation was detected between seropositivity and RT-PCR positive and negative cases. COVID-19 is spreading rapidly and there is a high percentage of asymptomatic carriers. The sensitivity of RT-PCR tests is not uniform and may not be able to detect all cases. On the other hand, serology can be used for large scale testing to detect the real extent to which the disease has spread.

Evaluation of IgM and IgG in COVID-19 Recovered Patients in Iraq.

Severe acute respiratory syndrome coronavirus-2 is a major threat to health care worldwide with high morbidity and mortality. Therefore, understanding the role of immune mechanisms and humoral response is vital in this disease. The present study aimed to investigate the relationship between Immunoglobulins (IgM, IgG) in COVID-19 recovered patients with age, gender, and severity of the disease. The duration of effect of antibody levels and protection against re-infection has also been evaluated in the patients. Three groups participated in this study; group 1: 0-14 days after recovery, group 2: 2 months after recovery, group 3: 3 months after recovery, group 4: 4-6 months after recovery, group 5: more than 6 months. The nasopharyngeal swab was used to confirm recovery by Real-Time Polymerase Chain Reaction (RT-PCR) technique. IgM and IgG antibody levels were evaluated using Enzyme-Linked Immuno Fluorescent Assay (ELIFA) technique. The results indicated that the IgM levels increased for one month during the seven days after infection and then decreased in most patients (P≤0.05). The mean of IgG in group 1 increased compared to those of other studied groups. A significant decrease was observed in group 2 compared to group 1, as well as in group 3 compared to groups 1, and 2. Also, a significant difference existed between group 4 compared to groups 1, 2, and 3. Finally, significant differences were noticed between group 5 compared to groups 1, 2, 3, and 4 (P≤0.05). No significant differences were observed in antibodies level between male, and female COVID-19 recovered patients in groups 1, 2, 3, 4, and 5 (P≤0.05). Finally, highly significant differences in IgG levels between mild, moderate, and severe subgroups in groups 1 and 2. The present study demonstrated that IgM and IgG against SARS-CoV-2 appeared in the early stages of the disease and decreased after 1 month and failed to maintain high levels during the 6-month observation.

Survey of rodent-borne pathogens in Singapore reveals the circulation of Leptospira spp., Seoul hantavirus, and Rickettsia typhi

Rodents living alongside humans increases the probability of encounter and also the transmission of rodent-borne diseases. Singapore’s cosmopolitan urban landscape provides a perfect setting to study the prevalence of four rodent-borne pathogens: Seoul hantavirus (SEOV), Leptospira species, Rickettsia typhi and Yersinia pestis, and identify the potential risk factors which may influence rodent density and transmission of rodent-borne diseases. A total of 1143 rodents were trapped from 10 unique landscape structures throughout Singapore. Real-time quantitative Polymerase Chain Reactions were used to detect pathogenic and intermediate Leptospira spp. and Yersinia pestis, whereas the seroprevalence of SEOV and R. typhi were analysed by Enzyme-Linked Immunosorbent Assay and Immunofluorescence Assay respectively. Multivariable logistic regression analysis was used to evaluate the association between prevalence of infection in rodent reservoirs and risk factors. Most of the rodents were caught in public residential developments (62.2%). Among the tested rodents, 42.4% were infected with Leptospira spp., while 35.5% and 32.2% were seropositive for SEOV and R. typhi respectively, whereas Yersinia pestis was not detected. Furthermore, risk factors including habitat, species, gender, and weight of rodents, influenced prevalence of infection to a varying extent. This study highlights the presence of Leptospira spp., SEOV and R. typhi in Singapore’s rodent population, suggesting the need for effective rodent management and sanitation strategies to prevent further circulation and transmission to humans.

Milk Tumor Necrosis Factor Alpha and Interleukin-1Beta Among Toxoplasma gondii-Free and Infected Women

Pro-inflammatory cytokines play an important role in intercellular communications. In the last two decades, many cytokines have been identified in human milk. These cytokines are variable according to different conditions such as pathogenic infections which strongly stimulated the immune response. The present study aims to determine of IL1β and TNF-α in Toxoplasma gondii-free and infected women in an attempt to clarify the impacts of the infections on cytokines especially in mother's milk. The serum and milk sample were collected from 96 samples (48 for seropositive and 48 for seronegative). To confirm the Toxoplasma gondii infection; enzyme linked immunofluorescence assay (ELIFA) was used to detect anti-Toxoplasma IgG antibodies. TNF-α and IL1β levels were measured by ELISA technique. Regarding milk TNF-alpha, there were statistical differences in Toxoplasma-infected from Toxoplasma-free subjects, whereas no statistical variations in TNF-α were found between other studied groups. IL1β showed significant variation just between milk and serum in infected and non-infected subjects, whereas no variations were recorded in other groups. In conclusion, based on statistical analysis, present study showed good evidence that toxoplasmosis is one of the risk factors for increasing milk cytokine. Also, a significant increase in TNF-α and IL1β in mother's breast milk may indicate a key role for inducing or developing of inflammations in suckling. Thus, further investigations are needed to cover this area of study.

Qualitative Method of Salmonella Screening, Identification and Confirmation in Shrimp Matrices using Loop-mediated Isothermal Amplification Method, Enzyme Linked Immuno Fluorescent Assay and Standard Conventional Method

Qualitative Method of Salmonella Screening, Identification and Confirmation in Shrimp Matrices using Loop-mediated Isothermal Amplification Method, Enzyme Linked Immuno Fluorescent Assay and Standard Conventional Method

Диагностический потенциал моноклональных антител к аденовирусу

The diagnostic potential of 4B7 and 6B12 monoclonal antibodies (mAbs) against human adenovirus hexon protein has been studied in various immunological tests, namely: in-cell enzyme-linked immunosorbent assay (cell-ELISA), sandwich ELISA, and immunofluorescent assay (IFA). It was shown that the sensitivity of cell-ELISA, sandwich ELISA and IFA was 96%, 86% and 84%, respectively as compared to PCR. Thus, 4B7 and 6B12 mAbs are promising immunoreagents for the construction of various diagnostic kits to use in laboratory practice for adenovirus detection in clinical samples. monoclonal antibodies, human adenovirus, adenovirus infection, diagnosis

Persistent Roseoloviruses Infection in Adult Patients with Epilepsy.

Background: Human herpesviruses (HHV)-6A, HHV-6B and HHV-7 are considered to be involved in the pathogenesis of epilepsy, a common neurological disorder. The objective of this study was to determine the association of roseoloviruses infection with epilepsy. Methods: 53 epilepsy patients and 104 ordinary blood donors were analyzed to determine presence of virus-specific antibodies by enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA), genomic sequences, viral load and gene expression by polymerase chain reactions (PCRs) and restriction analysis, HHV-6 protein expression by IFA and level of cytokines by ELISA. Results: Roseoloviruses genomic sequences in DNA samples from whole blood were found in 86.8% of patients versus 54.8% of controls and active infection was revealed only in patients with epilepsy (19.6% of roseolovirus-positive patients). Significantly higher viral load and more frequent gene expression was detected in patients compared to the controls. HHV-6-encoded protein expression was demonstrated in 53.3% of patients with previously detected HHV-6 DNA. Changes in level of cytokines were determined in patients with elevated viral load compared to the patients without elevated viral loads and to the controls. Conclusions: Results on frequent active HHV-6 and HHV-7 infection in epilepsy patient’ peripheral blood indicate on possible involvement of these viruses in the disease development.

Effect of heat treatment on activity of staphylococcal enterotoxins of type A, B, and C in milk

Intoxication by staphylococcal enterotoxins (SE) is among the most common causes of food-poisoning outbreaks resulting from the consumption of raw milk or products made thereof. The aim of our study was to analyze the thermal stability of SE and evaluate the inactivation of SE types A, B, and C (SEA, SEB, SEC) by autoclaving at 100°C, 110°C, and 121°C. Milk samples were inoculated with 38 Staphylococcus aureus strains that possessed the ability to produce SEA, SEB, or SEC and incubated at 37°C for 24 h. This incubation was followed by heat treatment at 100°C, 110°C, or 121°C for 3 min. Samples were analyzed by Staph. aureus plate count method on Baird-Parker agar and specifically for the presence of SE. An enzyme-linked immunofluorescent assay (ELFA) on a MiniVIDAS analyzer (bioMérieux, Marcy l'Étoile, France) was used to detect SE, which were determined semi-quantitatively based on test values. The obtained results were analyzed by means of nonparametric statistical methods. All samples (100%; 38/38) were SE-positive before heat treatment, and the positivity rates decreased after heat treatment at 100°C, 110°C, and 121°C to 36.8% (14/38), 34.2% (13/38), and 31.6% (12/38), respectively. The rates of positive samples differed between SEA, SEB, and SEC producers: SEA was detected in the highest amounts both before and after heat treatment. The amount of SE (expressed as test values) decreased significantly after heat treatment. Comparing amounts of SE in positive and negative samples before and after heat treatment, we can conclude that the success of SE inactivation depends on the amount present before heat treatment. The highest amount of SE and the highest rate of SE-positive samples after all heat treatments were found in samples with strains producing SEA. For SEB and SEC, lower amounts of enterotoxin were present and were inactivated at 100°C. Although temperatures of 100°C, 110°C, and 121°C may inactivate SE in milk, the key measures in prevention of staphylococcal enterotoxicosis are avoiding initial contamination of milk by Staph. aureus, promoting consumption of heat-treated milk, and preventing disruption of the cold chain during milk production and processing.

Seroprevalence and molecular characterization of Leishmania in dogs from an endemic area of zoonotic visceral leishmaniasis in Brazil

Visceral leishmaniasis (VL) can cause large-scale and tenacious epidemics with high fatality rates. Current seroprevalence and circulating Leishmania species were evaluated in dogs domiciled in the municipality of Sabará, a small historic and touristic city in the Brazilian state of Minas Gerais. A total of 3926 dogs domiciled in seven different districts of Sabará were serologically tested for canine visceral leishmaniasis (CVL) by indirect enzyme-linked immunosorbent (ELISA) and immunofluorescence (IFA) assays, in a two-years census survey (2011–2012). The average positivity rate of canine infection was 3.4%. Three additional diagnostic tests – imprint/smear direct parasitological, molecular (LnPCR) and myeloculture – were performed in a random sample of fifty seropositive dogs composed of symptomatic (39) and asymptomatic (eleven) animals. LnPCR showed 100% of positivity for Leishmania DNA in, at least, one among four tissue samples tested (mesenteric lymph node, skin, spleen and bone marrow), independently of the clinical canine group. Higher and statistically equivalent positivity rates (98% and 96%) for Leishmania DNA were found in canine lymph node and spleen. Asymptomatic dogs showed expressive positivity rates in all three additional diagnostic techniques. Leishmania infantum was confirmed as the etiological agent of CVL in Sabará.

Herpes Group Viruses: a Seroprevalence Study in Hemodialysis Patients.

Herpes group viruses (herpes simplex virus, HSV; varicella-zoster virus, VZV; cytomegalovirus, CMV; and Epstein-Barr virus, EBV) remain an important cause of morbidity in immunocompromised persons. The aim of the study was to analyze the prevalence of HSV-1, HSV-2, VZV, CMV and EBV in patients undergoing hemodialysis. During a three-year period (2013-2015), 152 consecutive serum samples from hemodialysis patients and 150 healthy subjects (control group) were tested for the presence of IgM/IgG antibodies to herpes group viruses. Serologic tests were performed using a commercial enzyme-linked immunosorbent assay (ELISA) or enzyme-linked immunofluorescent assay (ELFA). Hemodialysis patients showed significantly higher CMV IgG seropositivity compared to controls (88.2% vs. 78.7%, p=0.011). In addition, seroprevalence rates of HSV-1 and VZV were higher in hemodialysis patients; however, these differences did not reach statistical significance (85.5% vs. 80.0%, p=0.054 and 99.3% vs. 96.0%, p=0.051, respectively). The prevalence of HSV-2 and EBV was similar in both groups (12.5% vs. 12.7%, p=0.137 and 98.0% vs. 95.3%, p=0.113, respectively). There was no difference in IgG seropositivity according to gender and place of residence. Logistic regression showed that older age was a significant predictor for CMV and EBV IgG seropositivity (increase in age by one year: CMV OR=1.055; 95%CI=1.030-1.080 and EBV OR=1.075, 95%CI=1.023-1.130).

Short communication: Pasteurization as a means of inactivating staphylococcal enterotoxins A, B, and C in milk.

Our aim was to assess the effect of pasteurization temperature on inactivation of staphylococcal enterotoxins (SE). Milk samples were inoculated with log 4.38 to 5.18cfu/mL of 40 different Staphylococcus aureus strains having the ability to produce types A, B, or C SE and incubated at 37°C for 24h to develop SE. This incubation was followed by heat treatment for 15 s at 72, 85, and 92°C. Samples were analyzed for Staph. aureus count by plate method and, specifically, for SE presence. An enzyme-linked immunofluorescent assay on a MiniVIDAS analyzer (bioMérieux, Marcy l'Étoile, France) was used to detect SE, which were determined semiquantitatively based on test values. The Staph. aureus count in milk before pasteurization did not affect the amount of SE. Before pasteurization, SEB was detected in the lowest amount compared with other SE types. Staphylococcal enterotoxins were markedly reduced with pasteurization and inactivated at pasteurization temperatures to an extent depending on the amount in the sample before pasteurization. After pasteurization at 72°C, SE were detected in 87.5% of samples (35/40), after pasteurization at 85°C in 52.5% of samples (21/40), and after pasteurization at 92°C in 45.0% of samples (18/40). We determined that SE may still persist in milk even when Staph. aureus bacteria are inactivated through pasteurization. Although pasteurization may partially inactivate SE in milk, a key measure in the prevention of staphylococcal enterotoxicosis linked to pasteurized milk consumption is to avoid any cold chain disruption during milk production and processing.

The dysfunction of platelets in paroxysmal nocturnal hemoglobinuria

IntroductionThrombosis is a dangerous complication of paroxysmal nocturnal hemoglobinuria (PNH) and has a high mortality rate. However, the mechanism underlying the development of thrombosis in PNH remains unclear. To explore this, platelet function and serum complement activity were investigated in 14 patients with classical PNH, 11 with PNH aplastic anemia (AA) and 30 healthy controls. Material and methodsSerum concentrations of the terminal complement complex (sC5b-9) were determined by enzyme-linked immunofluorescence assay (ELISA), and the levels of C5b-9, CD61 and CD62p on platelet membranes were determined by flow cytometry. Clinical parameters were assessed, including D-dimer and platelet aggregation induced by adenosine diphosphate (ADP) and arachidonic acid (ARA). ResultsSerum sC5b-9 concentrations were significantly lower in the PNH/PNH-AA than in the control group (P<0.01). C5b-9 deposition was significantly higher on CD59− platelets than on CD59+ platelets in PNH/PNH-AA patients and healthy controls (P<0.01 for each). D-dimer concentration was significantly higher in PNH/PNH-AA patients – especially those with lactate dehydrogenase (LDH) concentrations>1000U/L – than in controls (P<0.05). CD61 (P<0.05) expression was lower on CD59+ platelets in PNH than in controls and CD5− platelets in PNH. Expression of CD62p (P<0.01) was lower on CD59− and CD59+ platelets (P<0.01) in PNH cases than in controls. Platelet aggregation stimulated by the agonists ADP and ARA in the PNH/PNH-AA patients was significantly lower than that in controls (P<0.05). ConclusionsThe adhesion and aggregation of platelets, especially of CD59+ platelets, were compensatively decreased in PNH/PNH-AA patients without active thrombosis.

Diagnostic Methods for CHIKV Based on Serological Tools.

This chapter presents the most commonly used serological methods for the diagnosis of Chikungunya virus (CHIKV) infection in humans. CHIKV is a mosquito-borne Alphavirus widely distributed in the tropical and subtropical regions of Africa, Asia, and America. CHIKV infection in human causes acute febrile illness frequently accompanied by severe joint pain. Most of the infected patients may develop chronic arthralgia that may persist for several months or years. Laboratory diagnosis of CHIKV infection is mainly based on molecular and serological tests. The serological tests represent a valuable tool for diagnosis and epidemiological studies. Enzyme-linked immunosorbent assay (ELISA) and immunofluorescence assay (IFA) are simple, rapid, and sensitive techniques widely used for the diagnosis of CHIKV infection. However, these methods represent a screening tool and often require confirmation by a second-line assays. Serum virus neutralization assay is more specific than ELISA and IFA tests and is considered a confirmatory test. Neutralization assay is employed to determine the titer of virus neutralizing antibodies against CHIKV in patients' sera. The basis of microneutralization assay (MNA), results interpretation, and procedures will be illustrated in this chapter.

American Tegumentary Leishmaniasis and Flow Cytometry: A Review

American Tegumentary Leishmaniasis (ATL) is a parasitic disease caused by protozoans of the genus Leishmania and transmitted by the bite of sandflies. It is considered neglected due to its high incidence and morbidity, mainly in developing countries. The treatment for this disease besides expensive, is toxic and has limitations such as resistance, both from the parasite and the host; and invasiveness, making it not quite acceptable for some patients. There is still no available vaccine for the disease, what makes the correct diagnosis the better alternative to guide the treatment and therefore manage it. The diagnosis of suspected cases has to gather epidemiological, clinical and laboratorial data, since the disease can be misdiagnosed with other dermatological conditions. Immunological methods are commonly used for detecting and monitoring diseases, and the main techniques used in the routine for ATL are the Montenegro Skin Test, Enzyme-linked Immunosorbent Assay, Immunofluorescence Assay and Western Blot. Although these approaches have good sensitivity levels, they lack on specificity and have some limitations like crossed-reactions with other diseases caused by trypanosomatid parasites. One different serological approach, which is becoming an alternative method for the diagnosis of this disease, is Flow Cytometry; it has shown balanced levels of specificity and sensitivity and also a better accuracy when compared to other methods used in routine. Therefore, this article shows the recent advances on the diagnosis of ATL by flow cytometry and by using the findings on the literature, aims to guide researchers on what should be the focus to have a better diagnostic method for this disease.

Potential Impact of Different Cytomegalovirus (CMV) IgM Assays on an Algorithm Requiring IgM Reactivity as a Criterion for Measuring CMV IgG Avidity

The measurement of cytomegalovirus (CMV) IgG avidity is a powerful tool for identifying individuals with recent CMV infection. Because such patients are expected to be positive for CMV IgM, several investigators have suggested that CMV IgG-positive sera first be screened for CMV IgM and then only the IgM-reactive sera be tested for avidity. We investigated the impact of different CMV IgM assays on such a reflexing algorithm using a panel of 369 consecutive IgG-positive serum samples submitted for avidity testing. A bead-based immunofluorescent assay (BIFA) identified 105 IgM-positive serum samples, whereas an IgM-capture enzyme immunoassay (EIA) identified 48 IgM-positive serum samples; this marked difference led us to evaluate additional CMV IgM assays. An enzyme-linked immunofluorescent assay (ELFA) and a chemiluminescent immunoassay (CIA) were used to test all sera with discordant BIFA/EIA results, all sera with concordant positive results, and selected sera with concordant negative results. The findings indicated that the ELFA would identify 74 CMV IgM-positive samples and the CIA would identify 64. Of the 23 low-avidity serum samples, 2 were IgM negative by BIFA, 3 by ELFA and CIA, and 4 by EIA; of the 23 intermediate-avidity serum samples, 6 were IgM negative by BIFA, 10 by ELFA, and 15 by EIA and CIA. In both these avidity groups, BIFA IgM-negative sera were also negative by the other 3 assays. These findings demonstrate that an algorithm requiring CMV IgM reactivity as a criterion for CMV IgG avidity testing does not identify all low-avidity sera and thus misses some cases of acute CMV infection.

Prospective study of cytomegalovirus seropositivity and risk of mortality from diabetes

Cytomegalovirus (CMV) infects 40% of the world population and has been suggested to be associated with diabetes; however, no prospective study has ever examined diabetes mortality associated with the infection. A cohort of 14,404 non-diabetic adult participants aged 17-90years from the Third National Health and Nutrition Examination Survey (1988-1994) was prospectively followed for mortality through 2006. CMV immunoglobulin G was measured by enzyme-linked immunosorbent assay and immunofluorescence assay. Diabetes death was assessed with death records from the National Death Index. Cox proportional hazards modeling was used to determine diabetes mortality risk associated with CMV infection, adjusting for socio-demographics, diabetes risk factors, and comorbidities. At baseline, 76.8% of subjects were CMV seropositive, and after an average follow-up of 13.7years, diabetes mortality rate per 10,000 person-years was 6.8 (95% CI 5.7, 8.0). Among seropositive participants, the diabetes death rate (8.4, 95% CI 7.0, 9.9) was more than four times the rate in seronegative ones (2.0, 95% CI 1.1, 3.6) (P value for the difference <0.001). In the adjusted Cox proportional hazards analysis, CMV seropositivity more than doubled the risk of diabetes mortality (HR 2.06, 95% CI 1.05, 4.06). CMV infection may thus predict future mortality from diabetes in non-diabetic people.

Potential beneficial effects of ixmyelocel-T in the treatment of atherosclerotic diseases

IntroductionAdvanced atherosclerotic lesions are characterized by lipid accumulation, inflammation, and defective efferocytosis. An ideal therapy should address all aspects of this multifactorial disease. Ixmyelocel-T therapy, an expanded autologous multicellular therapy showing clinical promise in the treatment of diseases associated with advanced atherosclerosis, includes a novel population of M2-like macrophages. Here, we examine the macrophages of ixmyelocel-T and determine their ability to influx modified cholesterol in an atheroprotective manner, maintaining cholesterol homeostasis and preventing cellular dysfunction and death, ultimately promoting reverse cholesterol efflux.MethodsApproximately 50 ml of whole bone marrow was obtained from healthy donors and shipped overnight. Bone marrow mononuclear cells (BMMNCs) were produced by using density gradient separation and cultured for approximately 12 days to generate ixmyelocel-T. CD14+ cells were isolated from ixmyelocel-T via positive selection for analysis. Ixmyelocel-T and human leukemia monocyte (THP-1) cells were loaded with acetylated low-density lipoprotein (Ac-LDL) for analysis. Flow cytometry and immunofluorescence were used to examine Ac-LDL uptake, expression of cytokines was analyzed by enzyme-linked immunofluorescence assay (ELISA), and quantitative real-time PCR was used to analyze expression of cholesterol-transport genes. Both the in vitro cholesterol efflux assay and in vivo reverse cholesterol transport assay were used to examine cholesterol transport.ResultsIxmyelocel-T macrophages take up acetylated low-density lipoprotein and express the scavenger receptors CD36 and scavenger receptor-B1 (SR-B1). Ixmyelocel-T did not become apoptotic or proinflammatory after lipid loading. The cholesterol transporter genes ABAC1 and ABCG1 were both statistically significantly upregulated when ixmyelocel-T macrophages were loaded with cholesterol. Ixmyelocel-T also exhibited enhanced apolipoprotein A-I (ApoAI)-mediated cholesterol efflux. In addition, in vivo reverse cholesterol-transport assay demonstrated that ixmyelocel-T was able to efflux cholesterol in this model.ConclusionsIxmyelocel-T macrophages influx modified cholesterol, remained anti-inflammatory in the face of lipid loading and inflammatory challenge, and displayed enhanced cholesterol efflux capabilities. These combined features suggest that this autologous multicellular therapy may exert beneficial effects in atherosclerotic diseases.

Clostridium difficile as a cause of acute diarrhea: a prospective study in a tertiary care center

Clostridium difficile-associated diarrhea (CDAD) is an increasing problem. Recent reports suggest presence of community acquired CDAD (CA CDAD). Studies in India have shown varied results. The following are the aims of this study: (a) the prevalence of CDAD and CA CDAD in patients with acute diarrhea; (b) the incremental yield of second stool sample for the diagnosis of C. difficile infection (CDI); and (c) the risk factors for CDI. Patients with acute diarrhea (<4 weeks) between April 2009 and December 2010 had two stool sample tested for C. difficile toxin (CDT) by enzyme-linked immunofluorescent assay. Demographic, clinical data, risk factors, clinical course, complications, treatment, and response were noted. Of 150 patients (mean age, 47.3 years; 76 males), 12 (8 %) had their first stool sample positive for CDT. Two patients (1.3 %) had community acquired CDI. The study group was compared with 138 patients ("control group", stool samples negative for CDT). Compared to the controls, the study group were more likely to have had intensive care unit (ICU) stay (p = 0.018) and tube feeding (p = 0.035). Eleven patients were treated with metronidazole. One patient did not respond to metronidazole and was treated with vancomycin. No patient developed complications of CDAD. The prevalence of CDAD in our population was 8 % and of CA CDAD was 1.3 %. There was no advantage of testing two samples. ICU stay and tube feeding were major risk factors for the CDAD. Metronidazole was an effective first-line therapy.