In vivo measurement of the surface energy of human fingernail plates.

The surface energy of the human nail plate is expected to influence the adhesion of microorganisms (and subsequent colonization and infections) as well as that of medicines (and subsequent drug permeation) and of cosmetics. The aim of the study was therefore to measure the surface energy of nail plates in vivo. The surface energy of healthy human fingernails (untreated, hydrated and abraded) and of hoof membranes (often used as a model for the nail plate) was estimated from contact angle measurements of liquids (water, formamide, diiodomethane and glycerol) on the nail plate and subsequent computation using the Lifshitz-van der Waals/acid-base (LW-AB) approach. The surface energy of untreated fingernail plates was found to be 34 mJ m(-2) . Most of this total energy was from the apolar Lifshitz-van der Waals component. When the polar component of the surface energy was analysed, the electron donor component was considerably larger than the electron acceptor one. Hydrating the nail plate had no significant influence on the surface energy. In contrast, abrasion caused a small, but statistically significant increase in the apolar surface energy component. The surface energy of bovine hoof membrane was similar to that of the fingernail plate. We conclude that the human fingernail plate is a low-energy surface and that bovine hoof membranes may be used as a substitute for the nail plate in certain experiments.

Immune durability and protection against SARS-CoV-2 re-infection in Syrian hamsters.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) has caused a pandemic. As immunity to endemic human coronaviruses (i.e. NL63 or OC43) wanes leading to re-infection, it was unknown if SARS-CoV-2 immunity would also decline permitting repeat infections. Recent case reports confirm previously infected individuals can become re-infected; however, re-infection may be due to heterogeneity in the initial infection or the host immune response, or may be the result of infection with a variant strain that escapes pre-existing immunity. To control these variables, we utilized the Syrian hamster model to evaluate the duration of immunity and susceptibility to re-infection with SARS-CoV-2. Hamsters were given a primary mock or SARS-CoV-2 infection (culture media or 105 TCID50 USA/WA1/2020 isolate, respectively). Mock and SARS-CoV-2 infected hamsters were then given a secondary SARS-CoV-2 infection at 1, 2, 4, or 6 months post-primary infection (n = 14/time point/group). After the primary SARS-CoV-2 infection, hamsters developed anti-spike protein IgG, IgA, and neutralizing antibodies, and these antibodies were maintained for at least 6 months. Upon secondary SARS-CoV-2 challenge, previously SARS-CoV-2 infected animals were protected from weight loss, while all previously mock-infected animals became infected and lost weight. Importantly, despite having high titres of antibodies, one SARS-CoV-2 infected animal re-challenged at 4 months had a breakthrough infection with replicating virus in the upper and lower respiratory tract. These studies demonstrate immunity to SARS-CoV-2 is maintained for 6 months; however, protection may be incomplete and, even in the presence of high antibody titres, previously infected hosts may become re-infected.

Intranasal SARS-CoV-2 RBD decorated nanoparticle vaccine enhances viral clearance in the Syrian hamster model.

Multiple vaccines have been developed and licensed for SARS-CoV-2. While these vaccines reduce disease severity, they do not prevent infection, and SARS-CoV-2 continues to spread and evolve. To prevent infection and limit transmission, vaccines must be developed that induce immunity in the respiratory tract. Therefore, we performed proof-of-principle vaccination studies with an intranasal nanoparticle vaccine against SARS-CoV-2. The vaccine candidate consisted of the self-assembling 60-subunit I3-01 protein scaffold covalently decorated with the SARS-CoV-2 receptor binding domain (RBD) using the SpyCatcher-SpyTag system. We verified the intended antigen display features by reconstructing the I3-01 scaffold to 3.4A using cryo-EM, and then demonstrated that the scaffold was highly saturated when grafted with RBD. Using this RBD-grafted SpyCage scaffold (RBD+SpyCage), we performed two unadjuvanted intranasal vaccination studies in the "gold-standard" preclinical Syrian hamster model. Hamsters received two vaccinations 28 days apart, and were then challenged 28 days post-boost with SARS-CoV-2. The initial study focused on assessing the immunogenicity of RBD+SpyCage, which indicated that vaccination of hamsters induced a non-neutralizing antibody response that enhanced viral clearance but did not prevent infection. In an expanded study, we demonstrated that covalent bonding of RBD to the scaffold was required to induce an antibody response. Consistent with the initial study, animals vaccinated with RBD+SpyCage more rapidly cleared SARS-CoV-2 from both the upper and lower respiratory tract. These findings demonstrate the intranasal SpyCage vaccine platform can induce protection against SARS-CoV-2 and, with additional modifications to improve immunogenicity, is a versatile platform for the development of intranasal vaccines targeting respiratory pathogens.

Annexin 5 mediates a peroxide-induced Ca(2+) influx in B cells.

Annexin 5 is a Ca(2+)-binding protein, the function of which is poorly understood. Structural and electrophysiological studies have shown that annexin 5 can mediate Ca(2+) fluxes across phospholipid membranes in vitro [1]. There is, however, no direct evidence for the existence of annexin 5 Ca(2+) channels in living cells. Here, we show that annexin 5 inserts into phospholipid vesicle membranes at neutral pH in the presence of peroxide. We then used targeted gene disruption to explore the role of annexin 5 in peroxide-induced Ca(2+) signaling in DT40 pre-B cells. DT40 clones lacking annexin 5 exhibited normal Ca(2+) responses to both thapsigargin and B-cell receptor stimulation, but lacked the sustained phase of the response to peroxide. This late phase was due to Ca(2+) influx from the extracellular space, demonstrating that annexin 5 mediates a peroxide-induced Ca(2+) influx. Thus, peroxide induces annexin 5 membrane insertion in vitro, and peroxide-induced Ca(2+) entry in vivo in DT40 cells requires annexin 5. Our results are consistent with a role for annexin 5 either as a Ca(2+) channel, or as a signaling intermediate in the peroxide-induced Ca(2+)-influx pathway.

Diagnosis of peste des petits ruminants infection in small ruminants through in-house developed Indirect ELISA: Practical considerations.

AIM: The work was conducted to diagnose peste des petits ruminants (PPR) outbreak through an in house developed indirect ELISA (thereafter referred as iELISA) its comparison with other available diagnostic tests and description of practical considerations in its development, utility and limitations. MATERIALS AND METHODS: An outbreak resembled to PPR occurred in two different places of southern Gujarat viz. Vapi and Navsari, affecting 622 animals, including both goat (n = 476) and sheep (n = 146). Animals displayed the typical signs of PPR at Vapi; however diarrhea was the inconsistent feature in animals of Navsari. The affection caused morbidity of 100% and mortality were 73.68% (n = 392/532) and 56.67% (n = 51/90) in Vapi and Navsari outbreaks, respectively. Relevant ante mortem and post mortem samples were collected from representative animals. At the outset of the epidemic no kit was available with us, so agar gel immunodiffusion (AGID) was carried out and a commercial ELISA (cELISA) kit was ordered for making diagnosis through antibody demonstration. Meanwhile, an iELISA was developed in house using PPR vaccine as antigen and protein G conjugated HRPO antibody as detector. Histopathology and results of sandwich ELISA were also used to diagnose PPR virus (PPRV) in the outbreak. RESULTS: The iELISA developed had detected PPRV antibodies in 22/24 samples (91.66%). Significant difference was observed in disease sensitivity pattern of two species by Chi-square test. While AGID failed to detect antibodies in any sample. Results were reconfirmed by comparing with commercially available cELISA kit. CONCLUSION: PPR is an economically important disease and for the rapid diagnosis of PPR the in house developed antibody capture iELISA can be a suitable cost effective alternative.

Presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate receptor-mediated stimulation of glutamate and GABA release in the rat striatum in vivo: a dual-label microdialysis study.

The existence of presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-type glutamate autoreceptors on glutamate nerve terminals in vitro has recently been demonstrated using synaptosomal and brain slice preparations. In the present study we have used a modification of a rapid dual-label intracerebral microdialysis method, previously developed by Young and co-workers(80,81) for the study of presynaptic mechanisms of neurotransmitter release, to investigate whether presynaptic AMPA receptors also play a role in the control of striatal glutamate release in vivo. For comparative purposes, the action of locally applied AMPA on striatal GABA release in vivo was also monitored. Local application of AMPA (0.01-100 microM), by reverse dialysis, into the striatum resulted in concentration-dependent increases in the Ca(2+)-dependent efflux of both [3H]L-glutamate and [14C]GABA. Maximum responses reached 142.0+/-6.5% and 166.8+/-7.7% of basal efflux for [3H]L-glutamate and [14C]GABA, respectively. No marked behavioural changes were observed at any dose of the agonist. Unexpectedly, the AMPA-evoked responses were not potentiated by the AMPA receptor desensitization inhibitors cyclothiazide (10-100microM) or aniracetam (1mM). Consistent with this finding, AMPA-stimulated [3H]L-glutamate and [14C]GABA efflux were significantly attenuated by co-perfusion with the selective, competitive AMPA receptor antagonist 6-nitro-7-sulphamoylbenzo(F)quinoxaline-2,3-dione (100microM) but not 1-(aminophenyl)-4-methyl-7,8-methylendioxy-5H-2,3-benzodiazepine (100microM), a non-competitive AMPA receptor antagonist known to interact with the cyclothiazide site to control AMPA receptor function. The broad spectrum ionotropic glutamate receptor antagonist, kynurenic acid (100-1000microM) also markedly inhibited the AMPA-evoked responses in the striatum in vivo. None of the antagonists, when given alone, influenced basal efflux of [3H]L-glutamate suggesting a lack of tonic regulatory control of glutamate release via presynaptic AMPA-type autoreceptors in the rat striatum. These results demonstrate the presence of presynaptic AMPA receptors, of a novel cyclothiazide- and aniracetam-insensitive subtype, on presynaptic nerve terminals in the rat striatum in vivo, acting to enhance glutamate and GABA release. Our data support the concept of AMPA receptor heterogeneity in vivo, a finding which may facilitate the development of novel, more selective drugs for the treatment of a range of neurological disorders associated with abnormal cerebral glutamate release. The pharmacological profile of these novel presynaptic receptors is currently under investigation.

Primary prostatic involvement in non-Hodgkin lymphoma.

We report 3 cases of primary extranodal lymphoma of the prostate, an unusual extranodal presentation rarely diagnosed antemortem. Symptoms of prostatism associated with an enlarged hard prostate with pyuria and hematuria in younger patients should suggest the diagnosis. Urine cytologic examination should aid in the diagnosis of this condition.

Evidence for a role of presynaptic AMPA receptors in the control of neuronal glutamate release in the rat forebrain.

The role of presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in controlling the neuronal release of excitatory amino acids has been investigated. Stimulation of presynaptic AMPA receptors by the endogenous agonist L-glutamate, or by (R,S)-AMPA, dose-dependently enhanced the Ca(2+)-dependent, tetrodotoxin-insensitive, electrically-stimulated release of [3H]D-aspartate from rat forebrain slices. This AMPA receptor-mediated response showed marked stereoselectivity with the activity residing solely in the (S)-isomer. (R)-AMPA was inactive in this respect. AMPA-evoked responses were significantly enhanced in the presence of the AMPA receptor desensitization inhibitor, cyclothiazide (10 microM). Moreover, responses to both AMPA and glutamate were inhibited by competitive (NBQX) and non-competitive (GYKI 52466) AMPA receptor-selective antagonists in a dose-dependent manner. These results provide strong support for the existence of presynaptic AMPA receptors acting to enhance the synaptic release of excitatory amino acids in the mammalian forebrain. Such a positive feedback system may play an important functional role in physiological (e.g., long-term potentiation) and/or pathological (e.g., epileptogenesis) processes in the mammalian central nervous system. AMPA-type autoreceptors may provide new targets for drug action.

Sports injuries in adolescents.

Musculoskeletal injuries are the most common injuries in youth sports. A number of growth-related factors unique to the adolescent age group play an important role in the epidemiology, types, and management of these injuries. These mainly relate to the physical and psychosocial growth and development during adolescence and are discussed in this article. Overuse injuries are more commonly seen in this age group in the primary care setting. Shoulder impingement, elbow and shoulder injuries related to throwing, stress injuries of the wrist, spondylolysis, and anterior knee pain are some of the overuse injuries reviewed in this article.

Thermodynamic proton-ligand and metal-ligand stability constants of some drugs.

The thermodynamic proton-ligand (pKa) and metal-ligand stability constants of clioquinol, clofibrate, nitrofurazone, and tetracycline with Cu2+, Zn2+, Mn2+, Mg2+, and Ca2+ have been determined at 35 degrees C in 50% ethanol-water media. An empirical pH correction for mixed-aqueous media has been applied. The metal-ligand stability constants were determined by following the Bjerrum Calvin titration technique as applied by Agrawal to mixed-aqueous solvents. The effect of the basicity of the ligand and the order of stability constants is discussed. The stability constants of the divalent metals follow the order: Cu2+ greater than Zn2+ greater than Mn2+ greater than Mg2+ greater than Ca2+ with all the drugs.

T lymphocyte subsets and antilymphocyte antibodies in lupus nephritis.

T lymphocyte subsets were determined in 12 patients with untreated systemic lupus erythematosus (SLE) and in 14 healthy controls. Six out of 8 (75%) patients with lupus nephritis had reduction in the percentage of T helper cells and low helper: suppressor cell ratios compared with controls. None of the 4 patients without nephritis had low ratios. Cold-reactive anti-lymphocyte antibodies cytotoxic to both the helper and the suppressor cells were detected in 7 of the 8 patients who had nephritis. Low T helper: suppressor cell ratio in SLE seems to correlate with the presence of active nephritis.

The rebellious adolescent. Evaluation and management of oppositional and conduct disorders.

A wide variety of management options are available to the primary care physician who is presented with a rebellious adolescent. After a careful assessment, the clinician and other health care professionals can choose a diverse combination of interventions: individual therapy, family therapy, youth-centered programs, community-centered programs, psychopharmacology, and others. Rebellious adolescents need access to comprehensive medical and mental health care, academic education (including sexuality education), and full employment opportunities. Primary care physicians can play a vital and sometimes pivotal role coordinating services and helping parents, school personnel, and therapists work with these youth. Even when dealing with the very difficult and resistant group of youth with CD and ODD, optimism for improvement should always be maintained by the clinician.

Spectrophotometric determination of clofibrate.

A sensitive method for the rapid and accurate determination of clofibrate by hydroxamic acid formation is described. Hydroxylamine hydrochloride is reacted with clofibrate in alkaline medium to give 4-chlorophenoxyisobutyrohydroxamic acid, which forms a purple complex with iron(III) in acidic medium. Common excipients do not interfere.

Smoking and children.

Tobacco use by children and adolescents is a major health threat. A number of carcinogens and other harmful compounds have been identified in tobacco smoke. The major component, nicotine, is highly addictive. In India, approximately 5500 children and adolescents start using tobacco products daily, some as young as 10 years old. The majority of users have first tried tobacco prior to age 18. Children and adolescents are exposed to the harmful effects of nicotine from smoking or second hand smoke from others; and from use of smokeless tobacco. There is increased prevalence of respiratory disease, ear and sinus infections, asthma, oral disease, and many long-term complications such as cardiovascular disease and cancers due to tobacco use. Prevention and treatment strategies include behavioural approaches and pharmacotherapy. There is an increased urgency especially, for countries like India to address the problem of tobacco use by children and adolescents as the tobacco industry faces legal and public opinion obstacles in Western countries like United States. The medical practitioner can play an important role by implementing the preventive and treatment strategies in his or her practice.

Treatment of attention-deficit hyperactivity disorder.

Attention-deficit hyperactivity disorder (ADHD) is a common disorder among children and adolescents with reported prevalence rates of between 3 and 10%. Recent reports suggest that a multimodal treatment approach is preferable to address many symptoms of ADHD and its associated problems for the children, the family, and the school. Stimulant medications remain the mainstay of treatment and are highly effective in more than 75% of patients. Improvement in the core symptoms of inattention, impulsivity, and hyperactivity are most noticeable. Recently approved extended release preparation of methylphenidate will allow once a day dosing. Reports of effectiveness of some non-stimulant medications such as bupropion, especially for adolescents, appear promising. A number of behavioral and psychosocial interventions can be used effectively as part of multimodal approach to address many ADHD-related problems. This article provides an update on practical information on the treatment of children and adolescents with ADHD who do not have other associated psychiatric disorders.

Recent advances in the hearing assessment of children.

The remarkable specificity and sensitivity of otoacoustic emissions (OAEs) in identifying cochlear dysfunction, and the speed and objectivity with which the test can be conducted has made the OAE procedure the 'standard-of-care' in pediatric audiology assessment. Together with the auditory brainstem responses (ABRs), the OAE procedure not only separates sensory from neural impairment, but also facilitates early audiologic diagnosis and management. This article describes some unique applications of the OAE procedure in the diagnosis, monitoring and treatment of auditory dysfunction.

Sports related mild traumatic brain injury in adolescents.

While head injuries are not common in youth sports, they may have catastrophic results. Concussion is also referred to as mild traumatic brain injury (MTBI). Although the occurrence of head injuries has been studied more extensively in American football, the findings have wider application for any MTBI. Recently, more attention has been directed at other sports in which both boys and girls participate. The diagnosis of MTBI is based on subjective findings and subtle changes in mental status. Other cerebral injuries requiring emergent or urgent neurosurgical attention should be ruled out. Objective tests such as CT scans and Magnetic Resonance Imaging (MRI) are usually without significant findings in MTBI. Neuropsychological testing may demonstrate areas of deficiencies, however, results may be difficult to interpret because of confounding factors. Complications following MTBI have been known to occur. The most catastrophic of these is second impact syndrome. There are a variety of guidelines for return to play following a concussion, which have been designed in an effort to avoid problems such as second impact syndrome. These guidelines are based more on clinical experience than on scientific evidence. Education, good training and coaching techniques, improved equipment, and rule changes and enforcement, can all help in curbing the sports related head injuries in adolescents.

Encopresis.

Encopresis is fecal soiling associated with functional constipation in a child. Constipation and encopresis are common problems in children. Encopresis is most common between ages 3 and 7 years. Infants and pre-school children present with a history of constipation and withholding maneuvers. The school-age child may have constipation and fecal soiling for some time prior to detection. In some children encopresis is associated with enuresis and urinary tract infection. Family education is the essential first step in management, followed by disimpaction of stool and complete evacuation of the rectum. Reaccumulation of stool should be prevented by appropriate use of laxatives and stool softeners. This is followed by a gradual weaning of the laxative regimen and instituting toilet training. Relapses may occur. Up to 50-60% of children achieve acceptable bowel control, free of soiling, within a year.

Substance abuse: a pediatric concern.

Use of alcohol, tobacco, and other drugs is a worldwide problem and affects many children and adolescents. The use of opiates, cannabis, tobacco, and alcohol have been well recognized for centuries in India. Abuse of alcohol is widespread in college campuses. In the United States, in 1997, 54.3% of high-school students had used an illicit drug by the time they entered 12th grade. In India the use of drugs and alcohol within the context of religious beliefs and local traditions has been historically documented. Children & adolescents are exposed to alcohol, cannabis products, and tobacco product. Several risk factors and protective factors have been identified. A screening psychosocial history is the most useful tool to identify non-specific indicators of substance abuse. In India the use of pan masala, and home brewed products containing marijuana and alcohol have special significance. Pan masala is shown to have carcinogenic, genotoxic, and clastogenic properties. The pediatrician can play a vital role in the appropriate recognition and early referral.