Evaluation of cytokine production by J774.G8 macrophages after treatment with biotherapics

Introduction: Strains of macrophages, such as murine J774.G8 macrophages, are susceptible to influenza A infection [1]. One of the responses to viral infection involves the production of various types of immunostimulatory cytokines by infected cells [2]. Methods: In the present study, the macrophage strain J774.G8, maintained in RPMI medium, was submitted to treatment with 10% V/V of two different biotherapics prepared from influenza H3N2, both at 30x. Additionally, two control groups were analyzed: macrophages stimulated with water 30x and macrophages without any treatment. Biotherapics were prepared from intact H3N2 influenza virus and H3N2 inactivated by alcohol 70%. The compounding of both biotherapics followed this procedure: one part of viral particles was diluted in 9 parts of sterile distilled water. The 1:10 sample was submitted to 100 mechanical succussions using Autic® Brazilian machine, originating the first dilution, named decimal (1x). 1 ml of this solution was diluted in 9 ml of solvent and was submitted to 100 succussions, generating biotherapic 2x. This procedure was successively repeated, according to Brazilian Homeopathic Pharmacopoeia, to obtain the biotherapic 30x. By the same technique, water vehicle was prepared in the potency of 30x to be used as control. All samples were prepared under sterile and aseptic conditions, using laminar flow cabinet, class II, and were stored in the refrigerator (8ºC), to avoid microbiological contamination. J774.G8 macrophages were stimulated for 2 days, in a total of six stimuli. Immediately before infection with 25 µl of H3N2 influenza virus, the supernatants were collected and frozen at -20 ºC for later analysis. Next, 24 hours after the virus infection, the supernatants were aliquoted and frozen under the same conditions. Three independent experiments were done in triplicate. Analysis of supernatants was performed by flow cytometry using the Mouse Inflammation Kit. The cytokines detected in this experiment were IL-10, IL 12, TNF-α and MCP1. Results: In all cases, there were no significant differences compared to control groups. However, the production of TNF-α detected in macrophages treated by intact and inactivated biotherapics presented a tendency to increase after infection. In fact, similar results were previously detected in other experiments conducted only with the intact biotherapic [3]. The release of the cytokine MCP1 in all experimental situations presented a tendency to decrease after the viral infection when compared to untreated macrophages. No statistically significant difference was detected in the production of IL 12 and IL 10. These experiments will be repeated to confirm the data obtained.

Evaluation of the genotoxic and mutagenic potentials of a living nosode compounded with Influenza A virus

Background: The influenza virus has been responsible for contagious respiratory diseases with high mortality rates [1]. Some drugs have been used to treat human influenza. However, these drugs cause many common side effects and induce the appearance of resistant viral strains [2]. The impact caused by the influenza virus has motivated the development of new approaches for the prevention and control of influenza [3]. Therefore, a new homeopathic medicine was developed using, as a starting point, the infectious influenza virus [4]. This belongs to a group called living nosodes [5]. However, its mutagenic and genotoxic potentials, especially when used in low dilutions, has not yet been evaluated and it is important because this biotherapic is prepared from living microorganisms. Different methods can be used to detect mutagenic and genotoxicic effects. Aims: This study aims to evaluate the genotoxic and mutagenic potentials of influenza A living nosode at different homeopathic potencies. Methodology: 1 ml of purified viral suspension was diluted in 9 ml of sterile distilled water. This sample was submitted to 100 mechanical succussions (approximately 3 Hz), using Autic® Brazilian machine, originating the first dilution, named decimal (1x). 1 ml of this solution was diluted in 9 ml of solvent and was submitted to 100 sucussions, generating biotherapic 2x. This procedure was successively repeated, according to Brazilian Homeopathic Pharmacopoeia, to obtain the biotherapic 30x [6]. By the same technique, water vehicle was prepared until 30x potency to be used as control. All samples were prepared in sterile and under aseptic conditions, using laminar flow cabinet, class II, and were stored in the refrigerator (8ºC). The samples 1x, 6x, 12x, 18x, 24x and 30x and water 30x (vehicle control) were analysed by: the Inductest, which assesses the ability of physical or chemical agents to promote lysogenic induction as a reflection of damage in DNA molecules in lysogenic bacteria, and the Ames test, which uses indicator strains of Salmonella typhimurium, sensitive to substances that can induce different types of mutation. Results: The Inductest showed no decrease in the survival fraction of the bacteria used, and no increase in the formation of lysogenic induction, in any tested potency. The same profile was obtained after the Ames test, with similar results to negative control. Conclusion: We can conclude that this living nosode compounded with Influenza A virus is not able to induce DNA damage in prokaryotic cells. This result permits us to conclude that patients who use this medicine have no side effects related to mutagenesis and genotoxicity.

Evaluation of the genotoxic and mutagenic potentials of homeopathic Candida albicans solutions

Background: Candida spp is naturally found in humans’ flora of skin, gastrointestinal and genitourinary tracts and, in general, up to 75% of the population does not have any symptom [1]. However, oral candidiasis is very common among HIV patients and patients undergoing chemotherapy. The treatment of oral candidiasis is necessary once the disease causes discomfort and dysphagia, resulting in poor nutrition, slow recovery, and prolonged hospital stay [2,3]. Preliminary results obtained by our group with a new biotherapic prepared from Candida albicans (Candida 30x) showed a great potential to reduce the candida yeast adhesion rate when the epithelial cells were pre-treated. This study is currently being developed with the evaluation of mutagenic and genotoxic potentials of several homeopathic solutions. Aims: The goal of this study was to assess the genotoxic and mutagenic potentials of different homeopathic potencies of C. albicans. Methodology: One part of C. albicans yeast obtained from Brazilian patient’s blood [4] was diluted in 9 parts of sterile water. This sample was submitted to 100 mechanical succussions (approximately 3 Hz), using Autic® Brazilian machine, originating the first dilution (1x). Then, 1 ml of this solution was diluted in 9 ml of solvent, submitted to 100 succussions, obtaining 2x potency. This procedure was successively repeated to obtain 30x potency, according to Brazilian Homeopathic Pharmacopoeia [5]. By the same technique, water vehicle was prepared until 30x to be used as control. All samples were prepared in sterile and aseptic conditions, using laminar flow cabinet, class II and were stored in the refrigerator (8ºC). The samples 1x, 6x, 12x, 18x, 24x and 30x of C. albicans and water 30x (vehicle control) were analysed by: the Inductest, which assesses the ability of physical or chemical agents to promote lysogenic induction as a reflection of damage in DNA molecules in lysogenic bacteria, and the Ames test, which uses indicator strains of Salmonella typhimurium, sensitive to substances that can induce different types of mutation. Results: In the Inductest no decrease in survival fraction of bacteria and no increase in the formation of lysogenic induction were detected independently of the homeopathic potency employed. The same profile was obtained after the Ames test, with similar results to negative control. Conclusion: Afterwards, we can conclude that these samples are not able to induce DNA damage in the cells tested. So, the use of this medicine does not present any side effects related to mutagenesis and genotoxicity.

Direct-Push Blood Transfusion Practice in the Neonatal Unit of a Tertiary Hospital in South-South, Nigeria

Introduction: Transfusion of blood is a life-saving intervention in the care of ill neonates. Donated blood is a scarce national resource and must be used in the most efficient way. Exchange blood transfusion using the blood bag is the commonest mode of blood delivery employed. Other modalities of safe and sustainable blood delivery should also be explored, especially where paucity of funds predominates. This study aims to assess the usefulness of the direct push method where applicable, as an alternative to blood bag delivery in neonatal units of resource poor settings. Methods: A two year retrospective study of newborns admitted in the neonatal wards of the University of Uyo Teaching Hospital. Data obtained were the age, gender, indication for admission, packed cell volume (PCV) before and after transfusion. Blood transfusion was done in aliquots over 24 hours under aseptic conditions, via a peripheral vein. The push and pull method was employed, with no anticoagulant in the syringe. Post-transfusion PCV was done at least 24 hours after the procedure. Results: Of the one thousand and seventy-seven (1077) admitted neonates, two hundred and thirty-nine (22.2%), received blood products. Of these, twenty-one (8.8%), received a direct whole blood transfusion. Age (days) of the neonates transfused ranged from 1 to 26 days, with a mean of 10.4 ± 8.13. The Packed Cell Volume (PCV) pre-transfusion ranged between 20% - 44%, with a mean of 30.05 ± 6.39 while post-transfusion PCV ranged between 31% to 51%, with a mean of 38.17 ± 5.52(Fig. 1). The commonest indication for transfusion was prematurity, 9(42.8%) and neonatal sepsis 5 (23.8%). Conclusion: The direct transfusion of blood occasionally used, seems a relatively safe practice to correct mild/moderate anaemia. It also provides sufficient blood, with the advantage of usage when the umbilical cord access is no longer feasible and where cost of blood would otherwise, hinder quick intervention. This practice may need further evaluation by other centers.

Alpha-diversity and microbial community structure of the male urinary microbiota depend on urine sampling method

Considerable variation exists in the methodology of urinary microbiota studies published so far including the cornerstone of any biomedical analysis: sample collection. The aim of this study was to compare the urinary microbiota of first-catch voided urine (FCU), mid-stream voided urine (MSU) and aseptically catheterised urine in men and define the most suitable urine sampling method. Forty-nine men (mean age 71.3 years) undergoing endoscopic urological procedures were enrolled in the study. Each of them contributed three samples: first-catch urine (FCU), mid-stream urine (MSU) and a catheterised urine sample. The samples were subjected to next-generation sequencing (NGS, n = 35) and expanded quantitative urine culture (EQUC, n = 31). Using NGS, Bacteroidetes, Firmicutes, and Proteobacteria were the most abundant phyla in our population. The most abundant genera (in order of relative abundance) included: Prevotella, Veillonella, Streptococcus, Porphyromonas, Campylobacter, Pseudomonas, Staphylococcus, Ezakiella, Escherichia and Dialister. Eighty-two of 105 samples were dominated by a single genus. FCU, MSU and catheterised urine samples differed significantly in three of five alpha-diversity measures (ANOVA, p < 0.05): estimated number of operational taxonomic units, Chao1 and abundance-based coverage estimators. Beta-diversity comparisons using the PIME method (Prevalence Interval for Microbiome Evaluation) resulted in clustering of urine samples according to the mode of sampling. EQUC detected cultivable bacteria in 30/31 (97%) FCU and 27/31 (87%) MSU samples. Only 4/31 (13%) of catheterised urine samples showed bacterial growth. Urine samples obtained by transurethral catheterisation under aseptic conditions seem to differ from spontaneously voided urine samples. Whether the added value of a more exact reflection of the bladder microbiota free from urethral contamination outweighs the invasiveness of urethral catheterisation remains to be determined.

Prevalence and Risk Factors for Unsuspected Spontaneous Ascitic Fluid Infection in Cirrhotics Undergoing Therapeutic Paracentesis in an Outpatient Clinic

Background and Aim: Ascites is a common complication of liver cirrhosis, making patients more vulnerable to infectious diseases such as spontaneous bacterial peritonitis. There hasn't been much research done on infectious ascitic fluid in asymptomatic patients. The purpose of the study was to find out the infectious asymptomatic ascitic fluid incidence and risk factors in liver cirrhotic patients. Materials and Methods: This cross-sectional study was conducted on 76 cirrhotic patients who underwent therapeutic paracentesis between September 2020 and February 2021 in an outpatient department of Jinnah Medical College, Peshawar. An 18-G catheter was used to collect ascitic fluid under strict aseptic conditions. Total and differential leucocyte counts, as well as total protein and albumin levels, were measured. The fluid was injected for bacterial culture of aerobic type and anaerobic blood culture bottles (10 mL each) under strict aseptic conditions. Individuals with abdominal pain, recent gastrointestinal bleeding, fever, SBP previous history, hepatic encephalopathy, impaired renal function, and treatment with antibodies were excluded. Written informed consent and ethical approval were taken prior to study conduction. Demographic details, liver disease severity, and etiology were noted along with laboratory technique-based biochemical tests, ascitic fluid count, and culture. SPSS version 20 was used for data analysis. Results: A total of 192 paracenteses were done on 76 liver cirrhosis patients with an average of 2.53 per patient. The overall mean age was 43.65±8.7 years. Of the total 76 patients, 55 (72.4%) were male and 21 (27.6%) were female. The ascites duration for study inclusion was 3 to12 months. Hepatitis B, fatty liver disease, hepatitis C, and drugs were the major causes of cirrhosis among study patients. The prevalence of Hepatitis B, fatty liver disease, hepatitis C, and drugs was 27 (35.5), 23 (30.3%), 11 (14.5%), and 15 (19.7%) respectively. The hepatic encephalopathy and variceal bleeding history were present in 16 (33.3%) and 32 (66.7%) respectively in a total of 48 (63.2%) cirrhosis patients. The class C and child Pugh class had 23 (30.3%) and 53 (69.7%) respectively. Null mortality was found in patients due to infection caused by spontaneous ascitic fluid. Conclusion: Our study found that hepatitis B, fatty liver disease, hepatitis C, and drugs were the major causes of cirrhosis. Asymptomatic ascitic fluid infection was extremely rare in cirrhotic patients who attended an outpatient clinic and underwent therapeutic paracentesis. Additionally, our study found that the peritoneal fluid asymptomatic spontaneous infection is rare among cirrhotic patients undergoing outpatient base therapeutic paracentesis. Further investigation for ascitic fluid analysis's role in such infection without treatment is to be carried out. Keywords: Ascitic fluid; Cirrhosis; Infection; Therapeutic paracentesis; Spontaneous bacterial peritonitis

The need for multiple vascular access or preemptive central venous catheterization in patients requiring planned interhospital helicopter transport: a flight doctor’s suggestion

Hemorrhagic shock can develop due to severe bleeding, such as after major trauma, postpartum or gastrointestinal bleeding. At least two peripheral intravenous routes with large-bore catheters are recommended to reverse hemorrhagic shock, and such functional intravenous routes are essential for the proper management of other concurrent diseases as well. Conditions during helicopter transportation are different from those seen in-hospital, and the primary concerns are to maintain aseptic conditions, protect patient’s privacy, and prevent infection risk, especially during pandemics, such as the ongoing COVID-19. Herein, I describe two recent experiences of improper management during helicopter transport due to intravenous line malfunction. Subsequently, based on my experience, I suggest the use of multiple intravenous routes or preemptive central catheterization in patients requiring helicopter transportation.

Incidence of Surgical Site Infection among Post C Section Women Presented in Surgical Emergency of tertiary care hospital

Aim: To rule out incidence and risk factors that are associated with infection at site of surgery after cesarean section. Method: A type of retrospective study was conducted on 100 women who underwent cesarean delivery procedures within a period of 8 month from September 2020 to April 2021 and presented in surgical emergency at Mayo Hospital Lahore. The socioeconomic, demographic and clinical parameters of patients were collected by a questionnaire form. A program known as SPSS version 20 was used for analysis of data that is collected in study. Result: After analyzing the data following factors are identified that causes surgical site infection: Higher BMI (more than 30kg/m2), loss of blood during cesarean section(more than 500mL), poor hygienic care after cesarean section, poor socioeconomic status leads to malnutrition of patient and that leads to poor wound healing and surgical site infection. Lack of education is also a key factor in SSIs. Cesarean done in emergency under improper aseptic conditions also promote SSIs. All these factors are associated with incidence of SSI. Conclusion: SSIS are conventional among women presented in surgical emergency of Mayo Hospital Lahore within 30 days of cesarean section. Management of risk factors causing SSIs in women after cesarean section may decrease the incidence of such infections. Keywords: Cesarean section, risk factors, infection at surgery site.

Rare presentation of Aspergillus as cystic swelling in pre-maxillary region

<p class="abstract">Subcutaneous fungal infections are caused by penetration of the causative fungi into the subcutaneous layer and usually present as single, localised non-tender nodular swelling. The diagnostic process is a vital dynamic process that requires effective communication and efficient collaboration. Aspergillus hyphae invade host tissues through release of various toxins like proteases, phospholipases, hemolysins, gliotoxin, aflatoxin, phthioic acid and other toxins. Under general anesthesia and proper aseptic conditions using Moure incision surgical debulking of the pre-maxillary mass was done and post-operatively oral antifungal medication was started. Multiple cystic swelling was sent for histopathological examination and found to be non-invasive Aspergillus fungal infection. Subcutaneous form of aspergillosis manifest as subcutaneous fungal infection. We presented an unusual case report of 45 years old immunocompetent female with cystic presentation of aspergillosis involving premaxillary region.</p>

The First Evidence of the Beneficial Effects of Se-Supplementation on In Vitro Cultivated Olive Tree Explants

Selenium is an essential micronutrient that provides important benefits to plants and humans. At proper concentrations, selenium increases plant growth, pollen vitality, the shelf life of fresh products, and seems to improve stress resistance; these effects can certainly be attributed to its direct and indirect antioxidant capacity. For these reasons, in the present work, the effects of selenium at different dosages on in vitro cultivated olive explants were investigated to observe possible positive effects (in terms of growth and vigor) on the proliferation phase. The work was carried out on four different olive cultivars: “San Felice”, “Canino”, “Frantoio”, and “Moraiolo”. The explants were cultured in aseptic conditions on olive medium (OM), with the addition of 4 mg·L−1 of zeatin, 30 g·L−1 of sucrose, and 7 g·L−1 of agar. The experimental scheme included a comparison between explants grown with five different concentrations of Na2SeO4 (0, 10, 20, 40, and 80 mg L−1) added to the medium during three successive subcultures. Interesting information has emerged from the results and all varieties responded to different concentrations of Selenium. The optimal Se dosages varied for each cultivar, but in general, Se concentration between 10 and 40 mg L−1 increased fresh and dry weight of the explants and shoot lengths. Se treatment induced in all cultivars and for all dosages used an increase in total Se content in proliferated explants. Furthermore, as the subcultures proceeded, the ability of the explants to absorb Se did not diminish. The Se content ranged from 8.55 to 114.21 µg kg−1 plant DW in ‘Frantoio’, from 9.83 to 94.85 µg kg−1 plant DW in ‘Moraiolo’, from 19.84 to 114.21 µg kg−1 plant DW in ‘Canino’, and from 20.97 to 95.54 µg kg−1 plant DW in ‘San Felice’. In general, the effect of selenium tends to decrease with the progress of subcultures and this suggests a sort of “adaptation” effect of the explants to its presence. The present study highlights for the first time the possibility of using in vitro cultures as biotechnological support to study supplementation with selenium and its effects on in vitro olive plant growth.