Correction: Ruchaya et al. Transplantation of Skeletal Muscle-Derived Sca-1+/PW1+/Pax7- Interstitial Cells (PICs) Improves Cardiac Function and Attenuates Remodeling in Mice Subjected to Myocardial Infarction. Cells2022, 11, 4050.

In the original publication [...].

Nelfinavir inhibition of Kaposi's sarcoma-associated herpesvirus protein expression and capsid assembly.

BACKGROUND: Antiviral therapies that target herpesviruses are clinically important. Nelfinavir is a protease inhibitor that targets the human immunodeficiency virus (HIV) aspartyl protease. Previous studies demonstrated that this drug could also inhibit Kaposi's sarcoma-associated herpesvirus (KSHV) production. Our laboratory demonstrated nelfinavir can effectively inhibit herpes simplex virus type 1 (HSV-1) replication. For HSV-1 we were able to determine that virus capsids were assembled and exited the nucleus but did not mature in the cytoplasm indicating the drug inhibited secondary envelopment of virions. METHODS: For KSHV, we recently derived a tractable cell culture system that allowed us to analyze the virus replication cycle in greater detail. We used this system to further define the stage at which nelfinavir inhibits KSHV replication. RESULTS: We discovered that nelfinavir inhibits KSHV extracellular virus production. This was seen when the drug was incubated with the cells for 3 days and when we pulsed the cells with the drug for 1-5 min. When KSHV infected cells exposed to the drug were examined using ultrastructural methods there was an absence of mature capsids in the nucleus indicating a defect in capsid assembly. Because nelfinavir influences the integrated stress response (ISR), we examined the expression of viral proteins in the presence of the drug. We observed that the expression of many were significantly changed in the presence of drug. The accumulation of the capsid triplex protein, ORF26, was markedly reduced. This is an essential protein required for herpesvirus capsid assembly. CONCLUSIONS: Our studies confirm that nelfinavir inhibits KSHV virion production by disrupting virus assembly and maturation. This is likely because of the effect of nelfinavir on the ISR and thus protein synthesis and accumulation of the essential triplex capsid protein, ORF26. Of interest is that inhibition requires only a short exposure to drug. The source of infectious virus in saliva has not been defined in detail but may well be lymphocytes or other cells in the oral mucosa. Thus, it might be that a "swish and spit" exposure rather than systemic administration would prevent virion production.

Expression and fusogenic activity of SARS CoV-2 Spike protein displayed in the HSV-1 Virion.

Severe acute respiratory syndrome coronavirus (SARS-CoV) is a zoonotic pathogen that can cause severe respiratory disease in humans. The new SARS-CoV-2 is the cause of the current global pandemic termed coronavirus disease 2019 (COVID-19) that has resulted in many millions of deaths world-wide. The virus is a member of the Betacoronavirus family, its genome is a positive strand RNA molecule that encodes for many genes which are required for virus genome replication as well as for structural proteins that are required for virion assembly and maturation. A key determinant of this virus is the Spike (S) protein embedded in the virion membrane and mediates attachment of the virus to the receptor (ACE2). This protein also is required for cell-cell fusion (syncytia) that is an important pathogenic determinant. We have developed a pseudotyped herpes simplex virus type 1 (HSV-1) recombinant virus expressing S protein in the virion envelop. This virus has also been modified to express a Venus fluorescent protein fusion to VP16, a virion protein of HSV-1. The virus expressing Spike can enter cells and generates large multi-nucleated syncytia which are evident by the Venus fluorescence. The HSV-1 recombinant virus is genetically stable and virus amplification can be easily done by infecting cells. This recombinant virus provides a reproducible platform for Spike function analysis and thus adds to the repertoire of pseudotyped viruses expressing Spike.

Return of the Tbx5; lineage-tracing reveals ventricular cardiomyocyte-like precursors in the injured adult mammalian heart.

The single curative measure for heart failure patients is a heart transplantation, which is limited due to a shortage of donors, the need for immunosuppression and economic costs. Therefore, there is an urgent unmet need for identifying cell populations capable of cardiac regeneration that we will be able to trace and monitor. Injury to the adult mammalian cardiac muscle, often leads to a heart attack through the irreversible loss of a large number of cardiomyocytes, due to an idle regenerative capability. Recent reports in zebrafish indicate that Tbx5a is a vital transcription factor for cardiomyocyte regeneration. Preclinical data underscore the cardioprotective role of Tbx5 upon heart failure. Data from our earlier murine developmental studies have identified a prominent unipotent Tbx5-expressing embryonic cardiac precursor cell population able to form cardiomyocytes, in vivo, in vitro and ex vivo. Using a developmental approach to an adult heart injury model and by employing a lineage-tracing mouse model as well as the use of single-cell RNA-seq technology, we identify a Tbx5-expressing ventricular cardiomyocyte-like precursor population, in the injured adult mammalian heart. The transcriptional profile of that precursor cell population is closer to that of neonatal than embryonic cardiomyocyte precursors. Tbx5, a cardinal cardiac development transcription factor, lies in the center of a ventricular adult precursor cell population, which seems to be affected by neurohormonal spatiotemporal cues. The identification of a Tbx5-specific cardiomyocyte precursor-like cell population, which is capable of dedifferentiating and potentially deploying a cardiomyocyte regenerative program, provides a clear target cell population for translationally-relevant heart interventional studies.

Sulfobutylether-ß-cyclodextrin: A functional biopolymer for drug delivery applications.

Sulfobutylether ß-cyclodextrin (SBE-ß-CD) is a polyanionic cyclic oligosaccharide that contains glucopyranose units forming a torus ring-like structure. SBE-ß-CD is gifted with many favorable properties viz. relatively high solubility (>50 folds compared to ß-CD), improved stability, and biocompatibility that praised SBE-ß-CD as a smart polymer for drug delivery applications. Commercially, SBE-ß-CD is popular by its brand name Captisol®. The present review discusses the structure, properties, and preparation methods of SBE-ß-CD-based inclusion complexes (ICs). Furthermore, we discuss here the preparation and applications of SBE-ß-CD ICs-based nanoparticulate drug delivery systems, which combines the merits of both, ICs (enhanced solubility) and nanoparticles (NPs, targeted therapy). Patents on and FDA-approved Captisol®-enabled products are tabulated in the benefit of readers. The toxicological aspects and current clinical status of SBE-ß-CD or SBE-ß-CD-based products are briefly explained in the present review. In our opinion, the present review would be a pathfinder to allow dissemination of information on SBE-ß-CD.

A Drosophila model of HPV16-induced cancer reveals conserved disease mechanism.

High-risk human papillomaviruses (HR-HPVs) cause almost all cervical cancers and a significant number of vaginal, vulvar, penile, anal, and oropharyngeal cancers. HPV16 and 18 are the most prevalent types among HR-HPVs and together cause more than 70% of all cervical cancers. Low vaccination rate and lack of molecularly-targeted therapeutics for primary therapy have led to a slow reduction in cervical cancer incidence and high mortality rate. Hence, creating new models of HPV-induced cancer that can facilitate understanding of the disease mechanism and identification of key cellular targets of HPV oncogenes are important for development of new interventions. Here in this study, we used the tissue-specific expression technique, Gal4-UAS, to establish the first Drosophila model of HPV16-induced cancer. Using this technique, we expressed HPV16 oncogenes E5, E6, E7 and the human E3 ligase (hUBE3A) specifically in the epithelia of Drosophila eye, which allows simple phenotype scoring without affecting the viability of the organism. We found that, as in human cells, hUBE3A is essential for cellular abnormalities caused by HPV16 oncogenes in flies. Several proteins targeted for degradation by HPV16 oncoproteins in human cells were also reduced in the Drosophila epithelial cells. Cell polarity and adhesion were compromised, resulting in impaired epithelial integrity. Cells did not differentiate to the specific cell types of ommatidia, but instead were transformed into neuron-like cells. These cells extended axon-like structures to connect to each other and exhibited malignant behavior, migrating away to distant sites. Our findings suggest that given the high conservation of genes and signaling pathways between humans and flies, the Drosophila model of HPV16- induced cancer could serve as an excellent model for understanding the disease mechanism and discovery of novel molecularly-targeted therapeutics.

Transplantation of Skeletal Muscle-Derived Sca-1+/PW1+/Pax7- Interstitial Cells (PICs) Improves Cardiac Function and Attenuates Remodeling in Mice Subjected to Myocardial Infarction.

We have previously shown that skeletal muscle-derived Sca-1+/PW1+/Pax7- interstitial cells (PICs) are multi-potent and enhance endogenous repair and regeneration. Here, we investigated the regenerative potential of PICs following intramyocardial transplantation in mice subjected to an acute myocardial infarction (MI). MI was induced through the ligation of the left anterior descending coronary artery in 8-week old male C57BL/6 mice. 5 × 105 eGFP-labelled PICs (MI + PICs; n = 7) or PBS (MI-PBS; n = 7) were injected intramyocardially into the border zone. Sham mice (n = 8) were not subjected to MI, or the transplantation of PICs or PBS. BrdU was administered via osmotic mini-pump for 14 days. Echocardiography was performed prior to surgery (baseline), and 1-, 3- and 6-weeks post-MI and PICs transplantation. Mice were sacrificed at 6 weeks post-MI + PICs transplantation, and heart sections were analysed for fibrosis, hypertrophy, engraftment, proliferation, and differentiation of PICs. A significant (p < 0.05) improvement in ejection fraction (EF) and fractional shortening was observed in the MI-PICs group, compared to MI + PBS group at 6-weeks post MI + PICs transplantation. Infarct size/fibrosis of the left ventricle significantly (p < 0.05) decreased in the MI-PICs group (14.0 ± 2.5%), compared to the MI-PBS group (32.8 ± 2.2%). Cardiomyocyte hypertrophy in the border zone significantly (p < 0.05) decreased in the MI-PICs group compared to the MI-PBS group (330.0 ± 28.5 µM2 vs. 543.5 ± 26.6 µm2), as did cardiomyocyte apoptosis (0.6 ± 0.9% MI-PICs vs. 2.8 ± 0.8% MI-PBS). The number of BrdU+ cardiomyocytes was significantly (p < 0.05) increased in the infarct/border zone of the MI-PICs group (7.0 ± 3.3%), compared to the MI-PBS group (1.7 ± 0.5%). The proliferation index (total BrdU+ cells) was significantly increased in the MI-PICs group compared to the MI-PBS group (27.0 ± 3.4% vs. 7.6 ± 1.0%). PICs expressed and secreted pro-survival and reparative growth factors, supporting a paracrine effect of PICs during recovery/remodeling. Skeletal muscle-derived PICs show significant reparative potential, attenuating cardiac remodelling following transplantation into the infarcted myocardium. PICs can be easily sourced from skeletal muscle and therefore show promise as a potential cell candidate for supporting the reparative and regenerative effects of cell therapies.

Progress on Thin Film Freezing Technology for Dry Powder Inhalation Formulations.

The surface drying process is an important technology in the pharmaceutical, biomedical, and food industries. The final stage of formulation development (i.e., the drying process) faces several challenges, and overall mastering depends on the end step. The advent of new emerging technologies paved the way for commercialization. Thin film freezing (TFF) is a new emerging freeze-drying technique available for various treatment modalities in drug delivery. TFF has now been used for the commercialization of pharmaceuticals, food, and biopharmaceutical products. The present review highlights the fundamentals of TFF along with modulated techniques used for drying pharmaceuticals and biopharmaceuticals. Furthermore, we have covered various therapeutic applications of TFF technology in the development of nanoformulations, dry powder for inhalations and vaccines. TFF holds promise in delivering therapeutics for lung diseases such as fungal infection, bacterial infection, lung dysfunction, and pneumonia.

Acute Rhabdomyolysis in a Child with Multiple Suspicious Gene Variants.

Rhabdomyolysis is diagnosed with creatinine kinase (CK) elevation beyond 1000 U/L or ten times above the normal upper limit. Severe episodes can be fatal from electrolyte imbalance, acute renal failure, and disseminated intravascular coagulation. A 13-month-old child was admitted with a CK of 82,090 U/L in the setting of respiratory tract infection-related hyperthermia of 106.9° farenheit. His medical history was significant for prematurity, dystonia, and recurrent rhabdomyolysis. His home medications clonazepam, clonidine, and baclofen were continued upon admission. He exhibited uncontrolled dystonia despite treatment for dystonia. Therefore, sedative infusions and forced alkaline diuresis were begun to prevent heme pigment-induced renal injury. Despite these interventions, his CK peaked at 145,920 U/L, which is rarely reported in this age group. The patient also developed pulmonary edema despite diuresis and required mechanical ventilation. Sedative infusions were not enough for dystonia management, and he needed the addition of a neuromuscular blocking infusion. He finally responded to these interventions, and the CK normalized after a month. He required a month of mechanical ventilation and two and a half months of hospitalization and extensive rehabilitation. We were able to avert renal replacement therapy despite pulmonary edema and an estimated glomerular filtration rate nadir of 21 mL/min/1.73 m2 based on the bedside Schwartz formula. He made a complete recovery and was discharged home. His growth and development were satisfactory for two years after that event. His extensive diagnostic workup was negative. Unfortunately, he died from septic and cardiogenic shock with mild rhabdomyolysis two years later. Prompt recognition, early institution of appropriate therapies, identification of underlying disease, and triggering events are pivotal in rhabdomyolysis management. Evidence-based guidelines are needed in this context.

A Rare Etiology for Ascending Paralysis in an Infant.

An 11-month-old male infant with ascending paralysis had an unremarkable initial cerebrospinal fluid (CSF) analysis and imaging. Progressive neurological symptoms resulted in repeated CSF sampling, microscopy, and plasma microbial cell-free DNA next-generation sequencing analysis, that in combination with epidemiology, confirmed the diagnosis.

Discovery and Anticancer Activity of Novel 1,3,4-Thiadiazole- and Aziridine-Based Indolin-2-ones via In Silico Design Followed by Supramolecular Green Synthesis.

Three crucial anticancer scaffolds, namely indolin-2-one, 1,3,4-thiadiazole, and aziridine, are explored to synthesize virtually screened target molecules based on the c-KIT kinase protein. The stem cell factor receptor c-KIT was selected as target because most U.S. FDA-approved receptor tyrosine kinase inhibitors bearing the indolin-2-one scaffold profoundly inhibit c-KIT. Molecular hybrids of indolin-2-one with 1,3,4-thiadiazole (IIIa-m) and aziridine (VIa and VIc) were afforded through a modified Schiff base green synthesis using ß-cyclodextrin-SO3H in water as a recyclable proton-donor catalyst. A computational study found that indolin-2,3-dione forms a supramolecular inclusion complex with ß-cyclodextrin-SO3H through noncovalent interactions. A molecular docking study of all the synthesized compounds was executed on the c-KIT kinase domain, and most compounds displayed binding affinities similar to that of Sunitinib. On the basis of the pharmacokinetic significance of the aryl thioether linkage in small molecules, 1,3,4-thiadiazole hybrids (IIIa-m) were extended to a new series of 3-((5-(phenylthio)-1,3,4-thiadiazol-2-yl)imino)indolin-2-ones (IVa-m) via thioetherification using bis(triphenylphosphine)palladium(II)dichloride as the catalyst for C-S bond formation. Target compounds were tested against NCI-60 human cancer cell lines for a single-dose concentration. Among all three series of indolin-2-ones, the majority of compounds demonstrated broad-spectrum activity toward various cancer cell lines. Compounds IVc and VIc were further evaluated for a five-dose anticancer study. Compound IVc showed a potent activity of IC50 = 1.47 µM against a panel of breast cancer cell lines, whereas compound VIc exhibited the highest inhibition for a panel of colon cancer cell lines at IC50 = 1.40 µM. In silico ADME property descriptors of all the target molecules are in an acceptable range. Machine learning algorithms were used to examine the metabolites and phase I and II regioselectivities of compounds IVc and VIc, and the results suggested that these two compounds could be potential leads for the treatment of cancer.

Significance of IL-7 and IL-7R in RA and autoimmunity.

While physiological levels of IL-7 are essential for T cell proliferation, survival and co-stimulation, its escalated concentration has been associated with autoimmune diseases such as Rheumatoid arthritis (RA). Expression of IL-7 and IL-7R in RA monocytes is linked to disease activity score and TNF transcription. TNF stimulation can modulate IL-7 secretion and IL-7R frequency in myeloid cells, however, only IL-7R transcription levels are downregulated in anti-TNF responsive patients. Elevated levels of IL-7 in RA synovial tissue and fluid are involved in attracting RA monocytes into the inflammatory joints and remodeling them into proinflammatory macrophages and mature osteoclasts. Further, IL-7 amplification of RA Th1 cell differentiation and IFNγ secretion, can directly prime myeloid IL-7R expression and thereby exacerbate IL-7-mediated joint inflammatory and erosive imprints. In parallel, IL-7 accentuates joint angiogenesis by expanding the production of proangiogenic factors from RA macrophages and endothelial cells. In preclinical models, blockade of IL-7 or IL-7R can effectively impair joint inflammation, osteoclast formation, and neovascularization primarily by impeding monocyte and endothelial cell infiltration as well as inhibition of pro-inflammatory macrophage and Th1/Th17 cell differentiation. In conclusion, disruption of IL-7/IL-7R signaling can uniquely intercept the crosstalk between RA myeloid and lymphoid cells in their ability to trigger neovascularization.

Mothball Ingestion in the Setting of G6PD Deficiency Causing Severe Hemolytic Anemia, Methemoglobinemia, and Multiple Organ Failure in a Toddler.

Mothballs containing naphthalene or paradichlorobenzene are known to cause hemolysis and methemoglobinemia. They can also affect the other organs, including the kidneys, liver, lungs, and skeletal muscles. The involvement of 1 or 2 organs at a time has been commonly reported. However, more than 2 organ dysfunction in mothball intoxication is rare and usually indicates severe illness. The intoxication can have more pronounced symptoms in children with glucose-6-phosphate dehydrogenase (G6PD) deficiency. We report this case of a previously healthy 13-month-old patient who presented with severe hemolysis, lactic acidosis, methemoglobinemia, acute renal failure, hepatic dysfunction, and rhabdomyolysis. He required aggressive fluid resuscitation, blood transfusions, and mechanical ventilation. The underlying etiology of his illness was initially unclear; however, upon repeated questioning, the father recalled the patient chewing on a mothball 3-4 days before admission. Hence, mothball intoxication was considered the most plausible clinical diagnosis in this patient. He was given N-acetylcysteine, instead of methylene blue, because of hepatic dysfunction and the fact that G6PD deficiency could not be ruled out in the presence of acute hemolysis. The patient made a full recovery after 2 weeks of intensive care unit management. G6PD testing after 3 months confirmed the deficiency. These mothballs are available in Hawai'i, but this is the first report of such a severe presentation to our knowledge. The presence of methemoglobinemia, severe hemolysis, and thorough history-taking helped us determine the diagnosis of mothball intoxication and enabled definitive treatment.

Synovial Fluid Cytokines, Chemokines and MMP Levels in Osteoarthritis Patients with Knee Pain Display a Profile Similar to Many Rheumatoid Arthritis Patients.

BACKGROUND: There are currently no effective disease-modifying drugs to prevent cartilage loss in osteoarthritis and synovial fluid is a potentially valuable source of biomarkers to understand the pathogenesis of different types of arthritis and identify drug responsiveness. The aim of this study was to compare the differences between SF cytokines and other proteins in patients with OA (n = 21) to those with RA (n = 27) and normal knees (n = 3). METHODS: SF was obtained using ultrasound (US) guidance and an external pneumatic compression device. RA patients were categorized as active (n = 20) or controlled (n = 7) based upon SF white blood cell counts (> or <300 cells/mm3). Samples were cryopreserved and analyzed by multiplex fluorescent bead assays (Luminex). Between-group differences of 16 separate biomarker proteins were identified using ANOVA on log10-transformed concentrations with p values adjusted for multiple testing. RESULTS: Only six biomarkers were significantly higher in SF from active RA compared to OA-TNF-α, IL-1-ß IL-7, MMP-1, MMP-2, and MMP-3. Only MMP-8 levels in RA patients correlated with SF WBC counts (p < 0.0001). Among OA patients, simultaneous SF IL-4, IL-6, IL-8, and IL-15 levels were higher than serum levels, whereas MMP-8, MMP-9, and IL-18 levels were higher in serum (p < 0.05). CONCLUSION: These results support the growing evidence that OA patients have a pro-inflammatory/catabolic SF environment. SF biomarker analysis using multiplex testing and US guidance may distinguish OA phenotypes and identify treatment options based upon targeted inflammatory pathways similar to patients with RA.

Plaquing of Herpes Simplex Viruses.

There are numerous published protocols for plaquing viruses, including references within primary literature for methodology. However, plaquing viruses can be difficult to perform, requiring focus on its specifications and refinement. It is an incredibly challenging method for new students to master, mainly because it requires meticulous attention to the most minute details. This demonstration of plaquing herpes simplex viruses should help those who have struggled with visualizing the method, especially its nuances, over the years. While this manuscript is based on the same principles of standard plaquing methodology, it differs in that it contains a detailed description of (1) how best to handle host cells to avoid disruption during the process, (2) a more useful viscous medium than agarose to limit the diffusion of virions, and (3) a simple fixation and staining procedure that produces reliably reproducible results. Furthermore, the accompanying video helps demonstrate the finer distinctions in the process, which are frequently missed when instructing others on conducting plaque assays.

Seropositivity of severe acute respiratory syndrome coronavirus 2 infection among healthcare workers of the Armed Forces medical services, India: A multicentric study.

BACKGROUND: Serosurveys provide the prevalence of infection and over time will reveal the trends. The present study was conducted to estimate the seroprevalence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) among healthcare workers (HCWs) and to analyse various characteristics (risk factors) associated with SARS CoV-2 infection. METHODS: Eight government designated Corona virus disease -19 (COVID-19) hospitals were selected based on the hospital admission of patients with COVID-19 and the local epidemiological situation in the region. Multistage population proportion to size sampling was performed for the selection of HCWs. Serosurvey was conducted using the enzyme-linked immunosorbent assay-based IgG antibody test (COVID KAVACH). Bivariate and multiple logistic regression was performed to find out the factor/factors associated with the positive antibody test. RESULTS: Out of 3255 HCWs that participated in the study, data of 3253 were analysed. The seroprevalence was 19.7% (95% confidence interval: 18.5-21.3%). Factors associated were location, category of HCWs, male sex, previously tested positive by the molecular test, training on infection prevention and control, personal protective measures, handwashing technique, close contact with a patient confirmed with COVID-19, use of personal protective equipment and symptoms in the last 30 days. However, in multiple logistic regression, only location, category, previously tested positive by the molecular test and symptoms in the last 30 days were statistically significant. CONCLUSION: HCWs are vulnerable to SARS-CoV-2 infection. One in five HCWs had detectable antibodies. The presence of antibodies among HCWs may help in their placement and triage. HCWs may be advised to report early in case of any symptoms of COVID-19. Preventive measures may be targeted based on the location, with particular emphasis on ancillary workers and nurses.

Sarcoidosis and autoimmunity.

PURPOSE OF REVIEW: Sarcoidosis is a poorly understood multisystem granulomatous disease that frequently involves the lungs but can affect any organ system. In this review, we summarize recent developments in the understanding of the immune dysregulation seen in sarcoidosis and propose a new expanded definition of human autoimmunity in sarcoidosis, and the implications it would have on treating sarcoidosis with targeted immunotherapy regimens in the future. RECENT FINDINGS: Sarcoidosis has been linked to infectious organisms like Mycobacterium and Cutibacterium, and certain manifestations of sarcoidosis have been linked to specific HLA alleles, but the overall pathogenesis remains uncertain. Sarcoidosis patients have similar patterns of cellular immune dysregulation seen in other autoimmune diseases like rheumatoid arthritis, and recent large-scale population studies show that sarcoidosis frequently presents with other autoimmune diseases. SUMMARY: Advancements in the understanding of sarcoidosis support its consideration as an autoimmune disease. Sarcoidosis patients carry a higher risk of comorbid autoimmune conditions which offers an excellent opportunity to further understand autoimmunity and explore biologic therapies in sarcoidosis treatment, and furthermore will better targeted immunotherapy regimens for sarcoidosis patients in the future.

Junctional Tachycardia as a Diagnostic Criterion in Acute Rheumatic Fever.

Acute rheumatic fever (ARF) is an acute inflammatory process resulting in rheumatic carditis, one of the most common acquired heart diseases in youth. Among the clinical manifestations of carditis, pathologic valve regurgitation and atrioventricular block are included in the criteria for the diagnosis of ARF. Besides atrioventricular block, ARF may often present with other arrhythmias, such as junctional tachycardia (JT). However, JT is currently not recognized as a criterion for the diagnosis of ARF. Three adolescents presented in our hospital with JT, polyarthralgia, and laboratory signs of inflammation with evidence of preceding group A Streptococcus infection. None of the patients fulfilled the diagnostic criteria of ARF. On the basis of the presumed diagnosis of ARF, all 3 patients were treated with intravenous steroids. Steroid therapy was given, and JT converted to sinus rhythm within an average of 62 hours. Subsequent electrocardiograms revealed variable degree of atrioventricular block in all 3 patients, providing clinical evidence and fulfilling the diagnostic criteria of ARF. Patients were monitored for a total 2 to 8 days before discharge on standard antiinflammatory treatment. Follow-up electrocardiograms and Holter monitoring revealed resolution of the atrioventricular block and lack of JT recurrence in all patients. On the basis of these sentinel cases, we propose that JT should be included as a diagnostic criterion for the diagnosis of ARF.