Three-dimensional imaging studies in mice identify cellular dynamics of skeletal muscle regeneration.

The function of many organs, including skeletal muscle, depends on their three-dimensional structure. Muscle regeneration therefore requires not only reestablishment of myofibers but also restoration of tissue architecture. Resident muscle stem cells (SCs) are essential for regeneration, but how SCs regenerate muscle architecture is largely unknown. We address this problem using genetic labeling of mouse SCs and whole-mount imaging to reconstruct, in three dimensions, muscle regeneration. Unexpectedly, we found that myofibers form via two distinct phases of fusion and the residual basement membrane of necrotic myofibers is critical for promoting fusion and orienting regenerated myofibers. Furthermore, the centralized myonuclei characteristic of regenerated myofibers are associated with myofibrillogenesis and endure months post injury. Finally, we elucidate two cellular mechanisms for the formation of branched myofibers, a pathology characteristic of diseased muscle. We provide a synthesis of the cellular events of regeneration and show that these differ from those used during development.

Plasma galectin-9 relates to cognitive performance and inflammation among adolescents with vertically acquired HIV.

OBJECTIVE: Adolescents with perinatally acquired HIV (AWH) are at an increased risk of poor cognitive development yet the underlying mechanisms remain unclear. Circulating galectin-9 (Gal-9) has been associated with increased inflammation and multimorbidity in adults with HIV despite antiretroviral therapy (ART); however, the relationship between Gal-9 in AWH and cognition remain unexplored. DESIGN: A cross-sectional study of two independent age-matched cohorts from India [AWH on ART ( n  = 15), ART-naive ( n  = 15), and adolescents without HIV (AWOH; n  = 10)] and Myanmar [AWH on ART ( n  = 54) and AWOH ( n  = 22)] were studied. Adolescents from Myanmar underwent standardized cognitive tests. METHODS: Plasma Gal-9 and soluble mediators were measured by immunoassays and cellular immune markers by flow cytometry. We used Mann-Whitney U tests to determine group-wise differences, Spearman's correlation for associations and machine learning to identify a classifier of cognitive status (impaired vs. unimpaired) built from clinical (age, sex, HIV status) and immunological markers. RESULTS: Gal-9 levels were elevated in ART-treated AWH compared with AWOH in both cohorts (all P  < 0.05). Higher Gal-9 in AWH correlated with increased levels of inflammatory mediators (sCD14, TNFα, MCP-1, IP-10, IL-10) and activated CD8 + T cells (all P  < 0.05). Irrespective of HIV status, higher Gal-9 levels correlated with lower cognitive test scores in multiple domains [verbal learning, visuospatial learning, memory, motor skills (all P  < 0.05)]. ML classification identified Gal-9, CTLA-4, HVEM, and TIM-3 as significant predictors of cognitive deficits in adolescents [mean area under the curve (AUC) = 0.837]. CONCLUSION: Our results highlight a potential role of Gal-9 as a biomarker of inflammation and cognitive health among adolescents with perinatally acquired HIV.

Pathogen and Antibody Identification in Children with Encephalitis in Myanmar.

OBJECTIVE: Prospective studies of encephalitis are rare in regions where encephalitis is prevalent, such as low middle-income Southeast Asian countries. We compared the diagnostic yield of local and advanced tests in cases of pediatric encephalitis in Myanmar. METHODS: Children with suspected subacute or acute encephalitis at Yangon Children's Hospital, Yangon, Myanmar, were prospectively recruited from 2016-2018. Cohort 1 (n = 65) had locally available diagnostic testing, whereas cohort 2 (n = 38) had advanced tests for autoantibodies (ie, cell-based assays, tissue immunostaining, studies with cultured neurons) and infections (ie, BioFire FilmArray multiplex Meningitis/Encephalitis multiplex PCR panel, metagenomic sequencing, and pan-viral serologic testing [VirScan] of cerebrospinal fluid). RESULTS: A total of 20 cases (13 in cohort 1 and 7 in cohort 2) were found to have illnesses other than encephalitis. Of the 52 remaining cases in cohort 1, 43 (83%) had presumed infectious encephalitis, of which 2 cases (4%) had a confirmed infectious etiology. Nine cases (17%) had presumed autoimmune encephalitis. Of the 31 cases in cohort 2, 23 (74%) had presumed infectious encephalitis, of which one (3%) had confirmed infectious etiology using local tests only, whereas 8 (26%) had presumed autoimmune encephalitis. Advanced tests confirmed an additional 10 (32%) infections, 4 (13%) possible infections, and 5 (16%) cases of N-methyl-D-aspartate receptor antibody encephalitis. INTERPRETATION: Pediatric encephalitis is prevalent in Myanmar, and advanced technologies increase identification of treatable infectious and autoimmune causes. Developing affordable advanced tests to use globally represents a high clinical and research priority to improve the diagnosis and prognosis of encephalitis. ANN NEUROL 2023;93:615-628.

Characterization of the biofilm matrix composition of psychrotrophic, meat spoilage pseudomonads.

Psychrotrophic Pseudomonas species are the key spoilage bacteria of aerobically stored chilled meat. These organisms readily form biofilms on meat under refrigerated conditions leading to consumer rejection and associated economic losses. Limited information is available on the matrix composition of the biofilms formed by these bacteria. We quantified and characterized the main components of the matrix of mono-species biofilms of selected Pseudomonas fragi and Pseudomonas lundensis strains using chemical analysis and Raman spectroscopy. The biofilms were grown at 10 °C and 25 °C on nitro-cellulose membranes placed on surface sterilized beef cuts. Extra-cellular polymeric substances of the matrix were extracted in soluble and bound forms and were chemically assessed for total carbohydrates, proteins and extra-cellular DNA. Both Pseudomonas species showed a significant increase in total carbohydrates and total proteins when grown at 10 °C as compared to 25 °C. Extra-cellular DNA did not show a strong correlation with growth temperature. Raman spectra were obtained from planktonic bacteria and membrane grown biofilms at 10 °C and 25 °C. Higher levels of guanine were detected in planktonic cells as compared to biofilm cells. This study suggests that psychrotrophic Pseudomonas species may respond to cold stress by increasing extra-cellular polymer secretions.

Vibrational spectroscopy combined with transcriptomic analysis for investigation of bacterial responses towards acid stress.

The ability of bacteria to tolerate acid stress plays an important role in their growth and survival. In particular, aciduric bacteria have several survival systems that prevent cell damage from acid stress. In this study, the effect of the bacterial stress induced by pre-adaptation at different pH values on the cellular macromolecules of Lactobacillus plantarum was investigated using Raman spectroscopy and Fourier transform infrared spectroscopy. The expression of key genes was also quantified to provide understanding of the transcriptional response of the cells to lethal acid stress conditions. Principal component analysis of the spectra exhibited marked differences in the spectral regions associated with carbohydrates, lipids, proteins, and nucleic acids for all acid-stressed cells compared to those of untreated control cells. The changes in spectroscopic and transcriptomic profiles that were observed revealed alterations in bacterial cell wall composition after acid treatment. The results suggest the existence of a complex bacterial stress response in which modifications of cellular compounds from pre-adaption at low pH are involved. This study demonstrates the potential application of vibrational spectroscopy techniques to discriminate between intact and injured bacterial cells as well as to study their stress responses after exposure to acid environments during food processing.

Effect of Drying Methods on Protein and DNA Conformation Changes in Lactobacillus rhamnosus GG Cells by Fourier Transform Infrared Spectroscopy.

Microencapsulation protects cells against environmental stress encountered during the production of probiotics, which are used as live microbial food ingredients. Freeze-drying and spray-drying are used in the preparation of powdered microencapsulated probiotics. This study examines the ability of Fourier transform infrared (FTIR) spectroscopy to detect differences in cells exposed to freeze-drying and spray-drying of encapsulated Lactobacillus rhamnosus GG cells. The FTIR analysis clearly demonstrated there were more significant molecular changes in lipid, fatty acid content, protein, and DNA conformation of nonencapsulated compared to encapsulated bacterial cells. The technique was also able to differentiate between spray-dried and freeze-dried cells. The results also revealed the extent of protection from a protein-carbohydrate-based encapsulant matrix on the cells depending on the type drying process. The extent of this protection to the dehydration stress was shown to be less in spray-dried cells than in freeze-dried cells. This suggests that FTIR could be used as a rapid, noninvasive, and real-time measurement technique to detect detrimental drying effects on cells.

First isolation of dengue virus from the 2010 epidemic in Nepal.

Dengue is an emerging disease in Nepal and was first observed as an outbreak in nine lowland districts in 2006. In 2010, however, a large epidemic of dengue occurred with 4,529 suspected and 917 serologically-confirmed cases and five deaths reported in government hospitals in Nepal. The collection of demographic information was performed along with an entomological survey and clinical evaluation of the patients. A total of 280 serum samples were collected from suspected dengue patients. These samples were subjected to routine laboratory investigations and IgM-capture ELISA for dengue serological identification, and 160 acute serum samples were used for virus isolation, RT-PCR, sequencing and phylogenetic analysis. The results showed that affected patients were predominately adults, and that 10% of the cases were classified as dengue haemorrhagic fever/ dengue shock syndrome. The genetic characterization of dengue viruses isolated from patients in four major outbreak areas of Nepal suggests that the DENV-1 strain was responsible for the 2010 epidemic. Entomological studies identified Aedes aegypti in all epidemic areas. All viruses belonged to a monophyletic single clade which is phylogenetically close to Indian viruses. The dengue epidemic started in the lowlands and expanded to the highland areas. To our knowledge, this is the first dengue isolation and genetic characterization reported from Nepal.

First Isolation of Dengue Virus from the 2010 Epidemic in Nepal

Dengue is an emerging disease in Nepal and was first observed as an outbreak in nine lowland districts in 2006. In 2010, however, a large epidemic of dengue occurred with 4,529 suspected and 917 serologically-confirmed cases and five deaths reported in government hospitals in Nepal. The collection of demographic information was performed along with an entomological survey and clinical evaluation of the patients. A total of 280 serum samples were collected from suspected dengue patients. These samples were subjected to routine laboratory investigations and IgM-capture ELISA for dengue serological identification, and 160 acute serum samples were used for virus isolation, RT-PCR, sequencing and phylogenetic analysis. The results showed that affected patients were predominately adults, and that 10% of the cases were classified as dengue haemorrhagic fever/ dengue shock syndrome. The genetic characterization of dengue viruses isolated from patients in four major outbreak areas of Nepal suggests that the DENV-1 strain was responsible for the 2010 epidemic. Entomological studies identified <i>Aedes aegypti</i> in all epidemic areas. All viruses belonged to a monophyletic single clade which is phylogenetically close to Indian viruses. The dengue epidemic started in the lowlands and expanded to the highland areas. To our knowledge, this is the first dengue isolation and genetic characterization reported from Nepal.

Patterns of injury in mycophenolate mofetil-related colitis.

Mycophenolate mofetil (MMF) was introduced as a new immune-suppression drug in the mid-1990s. It is widely utilized in solid-organ transplantation immune-suppression regimens. Side effects include gastrointestinal (GI) toxicity in the form of nausea, vomiting, and diarrhea. Physicians tend to reduce the dose of MMF or switch their patients to an enterio-coated formula to overcome the side effects. Because GI side effects are well linked to MMF, colonoscopy is not utilized in most of the cases to investigate the diarrhea. However, Crohn's disease-like changes in the colon, erosive enterocolitis, and graft versus host disease-like colonic changes associated with the use of MMF have been reported. Colonic findings in five patients whose symptoms resolved after substituting another agent for MMF are described in the present report. Repeat colonoscopy 4 months following discontinuation of MMF showed reparative changes in one of our patients. MMF is an important drug in organ transplantation immune-suppression regimens; however, with its widespread usage, additional side effects continue to be recognized.

Are autoimmune diseases or glomerulonephritis affecting the development of panel-reactive antibodies in candidates for renal transplantation?

Panel-reactive antibodies (PRA) are a major obstacle to kidney transplantation (KTx). It is not completely clear why only some patients develop PRA, whereas others do not. We hypothesized that other factors, such as autoimmune diseases involving the kidney, might be a trigger for PRA development. We reviewed the original diseases that led to renal failure and their possible role in PRA development. Charts of 270 patients on the active waiting list for KTx were reviewed for complete demographics, presence of PRA, peak PRA level, first KTx or retransplantation, original disease, blood transfusions, pregnancy and rejection. Patients were divided into group 1 (PRA >10%) and group 2 (PRA <10%). There was a significantly higher proportion of patients in group 1 with autoimmune diseases than in group 2. The same proportion was found significant for all of the patients as well as for the patients listed for the first KTx (new patients). Previous KTx has significant impact on both class I and II peak PRA levels when compared with new patients who are already sensitized. A subanalysis of retransplantation showed patients with autoimmune disease (54%) have more graft loss due to rejection compared with nonautoimmune disease (43%). There is an association between high PRA level and autoimmune diseases causing renal failure regardless of the previous KTx status. Besides the risk of recurrence, autoimmune disease seems to affect the risk of graft loss due to rejection.

Determination of immunoreactivity ofPlasmodium falciparum antigens, serum dilutions and biomaterials.

Immunoreactivity properties of serum dilutions andPlasmodium falciparum malaria antigens were measured and compared by ELISA technique using different ELISA plates to evaluate the role of antigens and serum dilutions for optimum binding. Also effort has been made to see the effect of reaction surface and material i.e. ELISA plates for binding capacity. Serological properties were estimated by ELISA methods for detection of malaria and determination of immunological characteristics. Three Pf antigens (PfAg) i.e. ring infected erythrocyte surface antigen: AR-1 (RESA), histidine-rich protein 2 antigen (HRP-2) and glycophospholipid antigen (grown and developed Pf antigen from PSJ-M strain): GPL1 have been used for serological testing of human blood samples by Enzyme Link Immunosorbant Assay (ELISA). 1∶100, 1∶1000 and 1∶10000 dilutions of Pf positive and negative serum (50 samples in each group) and 1∶1000 dilution of Pf antigens were used to measure immunoreactive properties by ELISA method. Result of PfAg-serum immunoreactivity study showed that GPL1 has the highest degree of immuno binding reactivity compared to other Pf antigens. HRP-2 and RESA antigens showed no significant difference to each other. Study also found that Costar and Fastec ELISA plates have a better Ag-Ab binding capability compared to immulon and Falcon plates at all dilutions of serum. Serum dilution of 1∶100 showed best binding and reactivity with Pf antigens followed by 1∶1000 and 1∶10000 showed lowest reactivity.

Design and development of an immunosensor for the detection of malaria in field conditions.

Eradication of malaria in Southeast Asian countries is still a distant goal, due to the absence of a simple, rapid and inexpensive diagnostic technique. Here, an immunosensor for the photometric detection of malaria, the malaria-detecting immunosensor (MDI), is developed to detect Plasmodium falciparum malarial antibodies in human blood. The method uses the principle of laser light-scattering by latex bead agglutinates in media monitored by a light-detecting device. Agglutination is induced by mixing antigen-coated latex beads with serum antibodies. Immunoreactions are measured in terms of the Tyndall effect in the transmitted beam detected by photodiodes. MDI sensitivity and specificity are compared with the results of enzyme-linked immunoabsorbent assay and laser light-scattering immunoassay techniques, which show that it is a good and sensitive monitoring device.

Testing of newly developed glycophospholipid antigen for the detection of P. falciparum malaria by laser light immunoassay in endemic and non-endemic areas.

A glycophospholipid (GPL) antigen isolated from Plasmodium falciparum culture supernatant has been tested for its antigenicity. Detection of malaria positive known blood samples and unknown field samples from endemic and non-endemic areas were compared. In this study laser light scattering immunoassay (LIA) was used for the detection of P. falciparum malaria. Test results of control (malaria negative samples from Surat) were compared with known positive samples and unknown malaria positive field samples. A positive correlation has been observed (97%) in falciparum positive samples from laboratory and unknown samples from endemic area (Haldwani) by LIA method using GPL antigen. From the results of the study it was found that GPL antigen has a better antigenic property and can detect almost all the cases of Pf malaria by LIA method.

Study of malaria in a village of lower Myanmar.

Malaria endemicity in lower Myanmar has been studied to identify the causes for the prevalence of malaria in Yeasitkan village of lower Myanmar. Vector mosquitoes were collected by mosquito net in cattlesheds and in human dwellings (indoor and outdoor) by biting and catching procedure for the identification of species, insecticide susceptibility test and sporozoites detection. Larvae of mosquitoes were also collected in and around the village for vector identification and for breeding sources. Malaria infection in humans was examined by blood examination and blood antibody detection by ELISA method. Results showed that malaria infection was 43.2% in children under 10 years of age and An. dirus and An. minimus were found as main vectors. Total parasite positive rate was found to be 41.28% and in this 78.87% were P. falciparum infections and remaining 18.31% were of P. vivax. Spleen positive rate has been found very high in children between 2 and 9 years (52.94%). Study indicates that villages near to dam areas are more prone to malaria infection.

Sensitivity and specificity of isolated antigen fromPlasmodium falciparum culture supernatant.

Immunological sensitivity and specificity properties of isolated Plasmodium falciparum (GPL) antigen from culture supernatant have been measured and compared with malarial antigens and non malarial filtered paper blood sera for potency and efficacy. Latex bead coded GPL, Pf and RESA antigens immunoreaction properties of human filter paper blood samples (FPB) were studied by laser light scattering immunoassay (LIA) and Enzyme linked immunoabsorbent assay (ELISA) techniques. Results of GP. antigen sensitivity study by LIA method showed a very high malaria antibody binding response (MABR) i.e. 6% compared with 78% with RESA and 88% Pf antigens. Malaria detection by ELISA method also found similar results. Specificity study of GPL antigen for different non malarial filter paper blood sera (NMFS) showed no immunoreaction however Pf and RESA antigen showed few positive immunological responses. These results suggest that sensitivity and specificity properties of isolated GPL antigen is better than other antigens.

Effect of serum dilution in diagnosis of malaria in community.

Recently, it has been shown that immunological methods can be used for the diagnosis of malaria other than sero-epidemiology. A study has been done to investigate optimum binding capacity of antigen-antibody (Ag-Ab) at different serum dilutions. For validating antigen-antibody (Ag-Ab) reaction at 1:100, 1:1000 and 1:1000 serum dilutions, have been tested in two different laboratories to establish validation of the ELISA method. Inter laboratory test on synthetic peptide (RI) ELISA was found comparable and meaningful for assessing malaria transmission in defined locality at 1:100 dilution. Results also showed that 1:1000 serum dilution can be useful for diagnostic purpose.

Well-breeding Anopheles dirus and their role in malaria transmission in Myanmar.

Mosquitos were collected with human and animal baits from March 1996 to January 1998 in four villages located along the Yadana gas pipe line in Yepyu township, Dawae district, Tanintharyi Division, southern Myanmar. A total of 23 anopheline species were collected. Anopheles dirus were abundant in pre-monsoon (May/June) an post-monsoon (October) months. All An. dirus caught both humans and cattle were assayed with specific, sporozoite enzyme-linked immunosorbent assays (ELISAs). A total of 5/250 (2%) caught with human bait was found positive with Plasmodium vivax from Eindayaza, Ohnbinkwin and Thaechaung during rainy and cool-dry months. Larval surveys also showed An. dirus larvae/pupae were caught from domestic wells (6 to 46% found positive). Clinical surveys indicated that transmission is hyperendemic and occur all year round in all four villages.

Hyperendemic malaria in a forested, hilly Myanmar village.

A 1-year longitudinal study of hyperendemic malaria was carried out at Tha-bye-wa village, Oktwin township, situated in the forested Bago mountain range in south-central Myanmar. Mosquito infectivity was assayed using specific, sporozoite enzyme-linked immunosorbent assays. Anopheles dirus was the predominant vector in the postmonsoon season (October); during the cool-dry season (January), both An. dirus and Anopheles minimus were vectors. Members of the Anopheles culicifacies complex were caught in the hot-dry season (April) but none was infective. The entomological inoculation rate was estimated to be at least 13.7 infective bites/person/year. Infective An. dirus were caught feeding on cattle as well as on humans. Three of the 4 positive An. dirus and both positive An. minimus were caught biting humans indoors in the second quarter of the night when most people were sleeping. This suggests that use of insecticide-impregnated bednets in this area could interrupt transmission.

The effect of tetanus toxoid in leprosy patients.

Since cases of lepra reaction following smallpox vaccination and BCG vaccination had been reported the effect of tetanus immunisation on leprosy patients (whether it may provoke a lepra reaction or not) was studied. Three doses of purified tetanus toxoid (one ml initially, one ml after six weeks and one ml after six months) were given to 357 leprosy patients and 60 patients living in the same environ were followed as controls. The antibody response following immunisation was followed in six lepromatous leprosy patients using toxin antitoxin neutralisation test at the Lf/1000 level in mice and in three of them the antibody titre of leprosy patients rose to satisfactory level. The number of lepra reactions in these patients was monitored for nine months (two months before vaccination, during the six months period of vaccination and one month after the last dose of vaccine). There was no significant rise in the number of patients with reaction following the vaccination.

Active immunization against tetanus III. Results of a five year study.

This is the first time in Burma that tetanus toxoids (purified and adsorbed) have been tested in over 250 non-immune adult volunteers and studied for a period of nearly five years. The safety and efficacy of these toxoids have been assessed by immunological, statistical and clinical methods. Both toxoids were found to be safe. The adsorbed toxoid was far superior to the fluid toxoid as an immunizing agent and optimum immunization regimens are proposed and presented.