Inflammatory responses to the novel coronavirus SARS-CoV-2, which causes COVID-19, range from asymptomatic to severe. Here we present a follow-up analysis of a longitudinal study characterizing COVID-19 immune responses from a father and son with distinctly different clinical courses. The father required a lengthy hospital stay for severe symptoms, whereas his son had mild symptoms and no fever yet tested positive for SARS-CoV-2 for 29 days. Father and son, as well as another unrelated COVID-19 patient, displayed a robust increase of SERPING1, the transcript encoding C1 esterase inhibitor (C1-INH). We further bolstered this finding by incorporating a serum proteomics dataset and found that serum C1-INH was consistently increased in COVID-19 patients. C1-INH is a central regulator of the contact and complement systems, potentially linking COVID-19 to complement hyperactivation, fibrin clot formation, and immune depression. Furthermore, despite distinct clinical cases, significant parallels were observed in transcripts involved in interferon and B cell signaling. As symptoms were resolving, widespread decreases were seen in immune-related transcripts to levels below those of healthy controls. Our study provides insight into the immune responses of likely millions of people with extremely mild symptoms who may not be aware of their infection with SARS-CoV-2 and implies a potential for long-lasting consequences that could contribute to reinfection risk.

Keywords: B cell; COVID-19; Complement; Interferon; MHC class II; SARS-CoV-2; SERPING1.

Hereditary angioedema is characterized by recurrent attacks of painless, non itching edema of the face and limbs and sometimes by abdominal symptoms. It is due to a deficiency of functional inhibitor of the first component of complement (C1 Inh). We present a case where a normal antigenic level of C1 Inh was found but no functional activity was present (B variant). A short review is given of the pathogenesis, heredity, diagnosis and treatment of the disease.

As described in the literature, hereditary angioedema (HAE) is an autosomal dominant disease that presents with recurrent events of angioedema caused by a) deficiency or b) functional alteration of the plasma protein C1 inhibitor (C1-inh); this enzyme is involved in the regulation of the complement, kallikrein-kinin, fibrinolytic, and coagulation systems. HAE is characterized by episodes of edema in the larynx, facial structures and tissues, gastrointestinal tract, or extremities. Laryngeal edema has been reported to occur predominantly after oral surgery. We describe the case of an 18-year-old Asiatic male, reporting an unremarkable medical history, who experienced complications following orthognathic surgery. Thirty hours post-op, the patient developed severe facial, pharyngeal, and glottic edema that compromised the airway, and an emergency tracheal intubation was performed. He was tested for C1-inh plasma levels, showing a sub-normal concentration and indicating a diagnosis of HAE. The patient received fresh-frozen plasma and improved throughout the day as his condition stabilized. Several cases of HAE following oral surgery have been reported, but, to the authors' knowledge, this is the first case reported following orthognathic surgery. This patient's treatment will be described, and a literature review of the disease and management methods will be provided.

Background: We aimed to describe the episodes and trends of admissions for community-acquired Respiratory Infections (RI) over a 12-year period and to assess the impact of Haemophilus influenza type b (Hib) vaccine on RI admissions in children aged up to 3 years.

Methods: We conducted a twelve-year retrospective observational study on all community-acquired RI admitted to Fattouma Bourguiba Hospital in Monastir Governorate (Tunisia) from 1 January 2002 to 31 December 2013. RI cases were selected from the Regional Registry of Hospital Morbidity. Data were coded according to ICD-10. To assess the impact of the Hib vaccine, three cohorts were defined based on vaccine status (unvaccinated cohort, first vaccinated cohort (VC) by monovalent form and second VC by pentavalent combination).

Results: Admissions for RI represented 17.6% (CI95%: 17.3-18.1) of all communicable diseases hospitalizations (n = 6 061/34 289). The crude incidence rates (CIR) per 100,000 inh were 24.2 for upper RI (URI) and 77.5 and for Lower RI (LRI) (p < 0.0001). Pneumonias represented 53.9% of LRI. Sex-ratio (male/female) was 1.12 for URI and 1.64 for LRI (p < 0.0001). At admission, the median age was 22 years (IQR: 3-52). Admission for Pneumonia increased significantly during study period (slope 'b' = 5.16; p < 0.0001) especially in children up to 5 years old (slope 'b' = 5.53) and in elderly (slope 'b' = 2.13). Among children up to 3 years old, the CIRs per 100,000 for Hib pneumonia admission were 11.6 in Non-Vaccinated Cohort (NVC), 10.6 in Vaccinated Cohort (VC) by protocol 1 (Hib Vaccine monovalent) and 0.80 in VC by protocol 2 (pentavalent vaccine combination).The relative risk reduction was 99% for protocol 2 (p < 0.001).

Conclusion: Admissions for RI in a tertiary level hospital were common with an increasing trend. The Hib immunization program, in particular the pentavalent combination, has had a positive impact on the reduction of related acute diseases.

Keywords: Hib vaccine; Respiratory Infection; Tunisia; burden; inpatients; pneumonia; trends.

The paper deals with a parietal frontal cerebral abscess caused by HITB biotype I in a girl aged 8 months. First a meningitis is suspected, then a tuberculous meningitis unsuccessfully treated with ampicillin, biseptol, respectively INH, rifampicin, pyrazinamide, prednisone, phenobarbital and chloramphenicol. The patient died through a central respiratory standstill on the 17th day of disease. The anatomopathological examinations revealed a giant parietal frontal cerebral abscess. H.influenzae, (serum type B, biotype I) resistant to ampicillin, chloramphenicol, Kanamycin, rifampicin and tetracycline but sensitive to erythromycin and neomycin was also found. A pharyngeal infection with HITB was presumably the origin of the abscess.

Acquired C1-inhibitor (C1-INH) deficiency is a rare and potentially life-threatening disorder, which presents with recurrent attacks of non-pitting oedema to the face, airway, limbs or gastrointestinal tract. It is often associated with underlying B-cell lymphoproliferative disorders. We describe a case of a 73-year-old man with acquired C1-INH deficiency who presented with nephrotic syndrome due to glomerular IgM deposition, secondary to an underlying secretory lymphoplasmacytic lymphoma. Both the acquired C1-INH deficiency and the nephrotic syndrome resolved when the underlying B-cell lymphoma was treated with rituximab and bendamustine, suggesting the underlying lymphoproliferative malignancy was driving both disorders.

This study aimed at developing double crosslinked isoniazid (INH)-loaded polymethyl-methacrylate-ethylcellulose (PMMA-EC) polyspheres for rate-controlled enteric drug delivery. A PMMA solution was manipulated with the addition of EC to produce polyspheres by drop-wise extrusion into a primary crosslinking solution of AlCl3 (25% w/v), before adding a second crosslinking solution of either 30% w/v BaCl2 (polysphere Batch A) or 30% w/v MgCl2 (polysphere Batch B). The polyspheres were then subjected to FTIR spectroscopic analysis, in vitro drug release studies, drug entrapment efficiency (DEE) determination as well as surface area and porositometric investigations. Molecular Mechanics (MM) simulations elucidated the interaction between the cations and the PMMA-EC combination. FTIR spectra revealed an affinity of PMMA for Ba(2+), Mg(2+) and Al(3+). SEM showed smooth robust polyspheres ranging between 4-6 mm. Porositometric analysis established that polysphere Batch A had larger pores (315.314 Åabs) than Batch B (234.603 Åabs). Drug release profiles from polysphere Batch A displayed burst release with 50% INH released within 2 h (N = 3) that was attributable to the larger ionic radius of the second crosslinker Ba(2+) compared Mg(2+) which was employed for polysphere Batch B. The latter produced polyspheres with superior control in INH release (<25% within 2 h) (N = 3) and a higher DEE with minimal pore formation. The experimental findings were well corroborated by the MM simulations.

A new method of determination of genetically predetermined type of biotransformation by acetylation rate using reversed-phase liquid chromatography (RP-HPLC) was described. The method is based on determination of isonicotinic hydrazide (INH) which is excreted with the patient's urine during 24 h period after oral administration of 0.4 g of the drug. INH is used as pharmacogenetic marker. Precolumn derivatization of 4-chloro-5,7-dinitrobenzofurazan is used at RP-HPLC determination of INH and a new drug phosphabenzide (diphenylphosphinylacetic hydrazide, DPPAH) with specrtophotometric detection in urine. The limit of INH detection was 0.27 mug ml(-1) and the one of DPPAH was 0.82 mug ml(-1). As a result of pharmacokinetic investigation it was discovered that bimodal distribution by acetylation rate for DPPAH is less apparent than in the case of INH. It is shown, that immunomodulator xymedone (N-(beta-oxyethyl)-4,6-dimethyldihydropirimidon-2) is the acetylation inductor of xenobiotics.

Materials with negative linear compressibility (NLC) counterintuitively expand along one specific direction coupled to the volume reduction when compressed uniformly. NLC with a large value is desired for compression and materials science. However, NLC is generally smaller than -20 TPa-1. High-pressure X-ray diffraction experiments reveal that the β-quartz-like InH(BDC)2 generates an extreme NLC (-62.4 TPa-1) by framework hinging. InH(BDC)2 is much safer and lower-cost than Au+/Ag+ and CN--containing materials that dominated the fields of large NLC. This work reconfirms that a negative thermal expansion flexible framework could likely exhibit large NLC. Moreover, a large NLC could be anticipated to arise from β-quartz-like or related frameworks composed of rigid linear ligands and flexible framework angles.

OBJECTIVE

To investigate the reproductive endocrine changes after inhba overexpression into rat ovary.

METHODS

Thirty Sprague-Dawley rats were randomly assigned to three groups. Inhba overexpression lentivirus vectors (LV-eGFP-inhba) were microinjected into rat ovary (INH group); Control animals received the same amount lentivirus vector empty (CON group) or LV-eGFP (GFP group). Antral follicle amount and diameter were counted and serum level of activin A, E2, P, FSH, and LH and the expression of ER-α, ER-β, PR, FSHR, and LHR were measured.

RESULTS

There was no significant difference among three groups in antral follicle amount; antral follicle diameter was increased in INH group rats compared with the other group rats. Serum levels of activin A, E2, P, and FSH were increased and LH was decreased in INH group rats compared with the other group rats. The mRNA and protein expression of ER-α, ER-β, FSHR was higher in INH group rats than that in the other group rats. There was no significant difference in mRNA and protein expression of PR among the three group rats, LHR expression was decreased in INH group rats compared with the other two group rats.

CONCLUSIONS

inhba overexpression in rat ovary in vivo may change reproductive endocrine function.

Complements in serum and middle ear effusion were determined in 20 patients with secretory otitis media, and compared with those in the normal controls. The C5 and C1-INH in serum of the patients were significantly higher. On the contrary, C9 and B factor (Bf) were significantly lower, and the circulatory immunocomplex was also higher. In patients with secretory otitis media, the middle ear effusion levels of C3, C4 and C5 were significantly lower, and Bf and immunocomplex were significantly higher than those in serum. The results suggest that the ability of complements in clearing immunocomplex is low. Therefore, the immunocomplex may deposit in the mucosa of the middle ear. Thus the permeability of capillary will be increased, and the middle ear effusion occurs.

OBJECTIVE

In this paper, effect of the Tolazoline as antagonist of the alpha-2 adrenergic receptors in patients with bronchial asthma and chronic obstructive bronchitis was studied, and also the effect of stimulation with Hexoprenaline of beta-2 adrenergic receptor after bronchi-constriction caused with Propranolol, and Acetylcholine.

METHODS

Lung function parameters are determined with Body plethysmography. In patients with bronchial asthma and chronic obstructive bronchitis was registered resistance (Raw), was determined the amount of intrathoracic gas volume (ITGV), and specific resistance was calculated as well (SRaw). Aerosolization was done with standard aerosolizing machine-Asema.

RESULTS

The study included a total of 21 patients. Two hours after the inhalation of Propranolol, in experimental group, it was applied the blocker of alpha-2 adrenergic receptors (Tolazoline 20 mg / ml with inhalator ways), which did not cause changes in bronchomotor tonus of tracheobronchial system (p>> 1.0). Meanwhile, at the same patient, stimulation of beta-2 adrenergic receptor with Hexoprenaline (2 inh x 0.2 mg) is associated with a significant decrease of the specific resistance of airways (SRaw, p < 0.01). Control group results show that after bronchi-constriction caused by Propranolol-aerosol (20 mg / ml) in patients with bronchial asthma and chronic obstructive bronchitis, an increase of specific resistance in airways was caused (SRaw, p < 0.01), which confirms the presence of hyper-reactive bronco-constrictor effects intermediated by vagal ways. Two hours after Propranolol, inhaled Hexorenaline has blocked the action of Propranolol, but not entirely. Furthermore, two hours after acetylcholine-aerosol (1 mg /ml) was applied, inhaled Ipratropium (2 inh x 1 mg) has fully blocked the action of chemical bronchoconstrictor mediators, causing a decline of specific resistance in the airways (SRaw; p < 0.01).

CONCLUSIONS

This suggests that primary mechanism, which would cause reaction in patients with increased bronchial reactibility, is prevalence of the cholinergic system over adrenergic one, and not the relationship in between alpha-2 and beta-2 adrenergic receptors.

BACKGROUND

The study represents the first investigation of outpatient use of beta lactam antibiotics in Montenegro carried out in accordance with internationally approved methodology (DDD/ATC).

OBJECTIVE

The objective of our study was to establish both the scope and overall use of beta lactam antibiotics, and to assess their compatibility with current pharmacotherapeutic guidelines and their use in developed countries.

METHODS

The retrospective pharmaco-epidemiological study comprised a 100%-sample of beta lactams that were used in the period prior to introduction of new reform strategy on drug market.

RESULTS

Beta lactam antibiotics (J01C, J01D) were the most frequently applied anti-infectives for systemic use (ATC group J) in 2000 (11.3 DDD/1000 inh./day, 61%). Penicillins (J01C) were the most utilized (8.0 DDD/1000 inh./day, 71%). Cephalosporin derivatives (cephalexin and cefaclor) accounted for the remaining 29% (3.3 DDD/1000 inh./day). Aminopenicillins were prevailing among penicillins (85%). Beta lactamase sensitive penicillins were in the second place and approximately accounted for 14%.

CONCLUSIONS

The results of our study showed that the use of beta lactam antibacterials could be estimated as partially satisfactory. There is a need to make additional efforts with a view of further rationalization.

In Alzheimer's disease, neurons slowly degenerate due to the accumulation of misfolded amyloid <em>β</em> and tau proteins. In our research, we performed extended studies directed at amyloid <em>β</em> and tau aggregation <em>inh</em>i<em>b</em>ition using <i>in cellulo</i> (<i>Escherichia coli</i> model of protein aggregation), <i>in silico</i>, and <i>in vitro</i> kinetic studies. We tested our li<em>b</em>rary of 1-<em>b</em>enzylamino-2-hydroxyalkyl multifunctional anti-Alzheimer's agents and identified very potent dual aggregation <em>inh</em>i<em>b</em>itors. Among the tested derivatives, we selected compound (<em>b</em>)18</<em>b</em>), which exhi<em>b</em>ited a unique profile of <em>b</em>iological activity. This compound was the most potent and <em>b</em>alanced dual aggregation <em>inh</em>i<em>b</em>itor (A<em>β</em><su<em>b</em>)42</su<em>b</em>) <em>inh</em>i<em>b</em>ition (<em>inh</em>.) 80.0%, tau <em>inh</em>. 68.3% in 10 μM), with previously reported <i>in vitro</i> <em>inh</em>i<em>b</em>itory activity against <i>h</i>BuChE, <i>h</i>BACE1, and A<em>β</em> (<i>h</i>BuChE IC<su<em>b</em>)50</su<em>b</em>) = 5.74 μM; <i>h</i>BACE1 IC<su<em>b</em>)50</su<em>b</em>) = 41.6 μM; A<em>β</em> aggregation (aggr.) <em>inh</em>. IC<su<em>b</em>)50</su<em>b</em>) = 3.09 μM). In docking studies for <em>b</em>oth proteins, we tried to explain the different structural requirements for the <em>inh</em>i<em>b</em>ition of A<em>β</em> vs tau. Moreover, docking and kinetic studies showed that compound (<em>b</em>)18</<em>b</em>) could <em>inh</em>i<em>b</em>it the amyloid aggregation process at several steps and also displayed disaggregating properties. These results may help to design the next generations of dual or selective aggregation <em>inh</em>i<em>b</em>itors.

Keywords: Alzheimer’s disease; aggregation inhibitors; molecular modeling; tau protein; β-Amyloid.

This research work explores the surface chemistry and drug-polymer interaction in the manufactured controlled release micro-particles. Isoniazid (INH) was used as a model anti-tubercular drug while Eudragit® S100 (S100), Eudragit® L100-55 based co-processed Acryl EZE (EZE) and Ethylcellulose ECN10 (ECN10) were used as polymeric carriers. INH containing micro-particles were prepared using a mini spray dryer B-290 (Buchi, Switzerland). The drug polymer ratios were optimized at 1:1 and 1:3 to evaluate the effect of polymers on the release of the drug from the micro-particles. Solid state characterization via SEM and particle size analysis of the manufactured micro-particles showed densely aggregated spherical particles with a mean diameter <10μm. The advanced surface analysis via EDS revealed a homogenous drug distribution on the spray dried micro-particles. The physico-chemical characterization carried out by using DSC and XRPD showed an increase in the amorphicity of the drug during the spray drying process while the chemical elemental analysis via XPS revealed a strong intermolecular interaction between the amine group of the drug and the carboxyl group of the polymers. As expected, the in vitro dissolution study showed a slow release pattern for the highly water soluble drug INH in acidic media (pH1.2) for the first 2h followed by a burst release upon changing the pH to 6.8. It was concluded that emerging spray drying processing can be used as a valuable tool to encapsulate drug for controlled release dosage forms by means of facilitating a possible drug/polymer interaction as outlined by novel XPS analysis.

(<em>b</em>)<em>B</em>ackground:</<em>b</em>) Apoptosis disruptions play su<em>b</em>stantial roles in pathogenesis of arthritis and its symptoms. Cytokines and their intracellular signaling have pivotal roles in arthritis pathophysiology. This study aimed to investigate the relationship <em>b</em>etween synovial Interleukin-6 (IL-6), nuclear factor kappa-<em>B</em> (NF-ĸ<em>B</em>), and fractalkine (FKN) in the changes of edema and apoptosis during adjuvantinduced knee arthritis. (<em>b</em>)Methods:</<em>b</em>) A total of 240 male Wistar rats were divided into different groups. Arthritis was evoked and the knee edema changes were evaluated <em>b</em>y Vernier caliper. Synovial IL-6 was assayed <em>b</em>y rat standard ELISA kit. Levels of NF-ĸ<em>B</em>, fractalkine, and apoptotic indicators in the synovium were evaluated <em>b</em>y Western <em>b</em>lot method. Results were expressed as Mean± SEM. To analyze within-group variations, repeated measures ANOVA, followed <em>b</em>y post hoc Tukey's test was used (SPSS, 16). Independent samples t test was used to designate significant differences in knee diameter, synovial level of IL-6, apoptotic markers, NF-ĸ<em>B</em>, and FKN <em>b</em>etween groups. Significance level was set at P≤ 0.05. (<em>b</em>)Results:</<em>b</em>) The injection of Complete Freund's Adjuvant (CFA) caused intense knee edema (P< 0.001), which was reduced <em>b</em>y implementing anti-IL-6 (P< 0.001), anti-FKN (P< 0.001), <em>Inh</em>-NF-ĸ<em>B</em> (P< 0.001), and anti-FKN+ <em>Inh</em> NF-k<em>B</em> (P< 0.001). The results indicated elevated levels of apoptotic markers during the acute phase (P = 0.010), along with an increase in IL-6 (P< 0.001), NF-ĸ<em>B</em> (P< 0.001), and FKN (P= 0.030). Although IL-6 (P< 0.001), NF-ĸ<em>B</em> (P= 0.001), and FKN (P= 0.007) levels elevation continued during the chronic phase, the apoptosis markers decreased in this phase (P= 0.050). The findings revealed that Anti-IL-6 treatment during different phases of the study could change the synovial NF-ĸ<em>B</em> and FKN. (<em>b</em>)Conclusion:</<em>b</em>) It seems that time-dependent variations in apoptotic markers level may <em>b</em>e involved in pathogenesis of adjuvant-induced knee arthritis. In conclusion, synovial IL-6 through NF-ĸ<em>B</em>- FKN pathway can play an important role in this process.

Keywords: Adjuvant; Apoptosis; Fractalkine; IL-6; Inflammation; NF-ĸB.

OBJECTIVE

Analyze the decision to enact or to refrain from chemoprophylaxis (CP) with isoniazide (INH) in patients who are intravenous drug users (IVDU) in Spain infected by the human immunodeficiency virus (HIV), either classified or not with hypersensitive skin tests.

METHODS

With the bibliographic information available and the help of decision tree, an analysis of the effectiveness and of the consequences of drug costs of CP with INH in those patients is performed.

RESULTS

Overall, the HIV+ IVDU benefit from CP is an increase in survival of 80 days, with a savings of 7,252 pesetas per patient. The intradermal reaction with PPD and the retarded hypersensitivity tests (HCR) allow us to classify them in three subgroups: a) PPD+ where CP is universally admitted and thus corroborates our study; b) PPD-/HCR- where CP increases survival 201 days and saves 20,616 pesetas per patient; and c) PPD-/HCR+ where survival is increased 33 days and the pharmacological costs increase 1,536 pesetas per patient under CP.

CONCLUSIONS

For the present situation in Spain, CP with INH is effective in HIV+ IVDU patients, independent of the results of the intradermal reaction skin tests.

<A<em>b</em>stractText>Hereditary angioedema (HAE) may <em>b</em>e fatal and diagnosis can <em>b</em>e delayed up to 10 years. We aimed to screen HAE in two villages <em>b</em>ased on an index case of HAE and to investigate for the mutation of the C1 esterase inhi<em>b</em>itor (C1-<em>INH</em>) gene.</A<em>b</em>stractText><A<em>b</em>stractText>A total of 124 people were screened in two villages. The frequency and severity of symptoms were scored. C4, C1-<em>INH</em> levels and C1-<em>INH</em> activity were measured. We investigated for mutations of the C1-<em>INH</em> gene.</A<em>b</em>stractText><p><div>(<em>b</em>)Results</<em>b</em>)</div>Thirty-five cases of type I HAE and 7 cases of type II HAE were determined. Thirty-one (73.8%) patients diagnosed with HAE were 18 years old or younger. There was a positive correlation <em>b</em>etween C4 levels, C1-<em>INH</em> levels (<i>p</i> < 0.0001, <i>r</i> = 0.81), and C1-<em>INH</em> activity levels (<i>p</i> < 0.0001, <i>r</i> = 0.631) and <em>b</em>etween the age at diagnosis and severity score (<i>p</i> < 0.0001, <i>r</i> = 0.651). A positive correlation was found <em>b</em>etween the age at first symptom onset and C4 levels (<i>p</i> = 0.002, <i>r</i> = 0.774), and C1-<em>INH</em> levels (<i>p</i> = 0.006, <i>r</i> = 0.714). A marginally significant negative correlation was found <em>b</em>etween C1-<em>INH</em> activity levels and severity scores (<i>p</i> = 0.1, <i>r</i> = -0.515). We identified a novel heterozygous 1033G>T missense variant of the C1-<em>INH</em> gene, SERPING1, in patients with type I HAE.</p><A<em>b</em>stractText>There are long delay periods in the diagnosis of HAE and when the index case is present, family screening may <em>b</em>e very important and even life-saving, in particular, in paediatric patients without symptoms. Furthermore, the present study provides evidence to link a novel mutation, c.1033G>T, to the development of HAE in a large HAE family from Turkey.</A<em>b</em>stractText>

<p><div>(<em>b</em>)BACKGROUND</<em>b</em>)</div>Immune-mediated hemolytic anemia (IMHA) is a common disease that affects all <em>b</em>reeds of dogs and is associated with significant mor<em>b</em>idity and mortality. Intravascular hemolysis of erythrocytes in IMHA is caused <em>b</em>y complement activation and is often fatal. No current treatments target complement activation in canine IMHA. Human C<su<em>b</em>)1</su<em>b</em>) esterase (C<su<em>b</em>)1</su<em>b</em>)-<em>INH</em>) reduces canine complement-mediated hemolysis in vitro, and a recent pharmacokinetic analysis of an FDA licensed formulation of C<su<em>b</em>)1</su<em>b</em>)-<em>INH</em> in dogs confirmed that a 50 IU/kg dose of C<su<em>b</em>)1</su<em>b</em>)-<em>INH</em> is safe to administer to dogs, and effectively inhi<em>b</em>its canine complement mediated hemolysis ex-vivo. The C<su<em>b</em>)1</su<em>b</em>)INCH randomized controlled trial will evaluate the efficacy of this drug in dogs with intravascular hemolysis.</p><p><div>(<em>b</em>)METHODS</<em>b</em>)</div>We will conduct a multicenter, place<em>b</em>o-controlled dou<em>b</em>le-<em>b</em>lind randomized clinical trial of C<su<em>b</em>)1</su<em>b</em>)-<em>INH</em> in dogs with intravascular hemolysis due to IMHA. We will randomize 18 dogs to receive three doses of intravenous C<su<em>b</em>)1</su<em>b</em>)-<em>INH</em> or saline in 24 h. Immunosuppressive and antithrom<em>b</em>otic therapies will <em>b</em>e standardized. Primary outcome measures will <em>b</em>e changes in plasma free hemoglo<em>b</em>in, serum concentrations of LDH, <em>b</em>iliru<em>b</em>in, and haptoglo<em>b</em>in. Using patient samples, we will evaluate complement activation in canine IMHA using a novel C5<em>b</em>-9 ELISA assay, flow cytometric detection of C3<em>b</em> on RBC, and <em>b</em>y measurement of residual plasma complement activity. Secondary outcome measures will <em>b</em>e survival to hospital discharge, duration of hospitalization, num<em>b</em>er and volume of red <em>b</em>lood cell transfusions, and rescue therapy requirements. We will monitor dogs for adverse drug reactions. Sample size was estimated from pilot data on LDH and hemolysis index (HI) in dogs with IMHA. To detect 2-way differences <em>b</em>etween the upper and lower 50% of the LDH and HI values of equivalent size with 80% power at P < 0.05 will require 9 dogs in each arm.</p><A<em>b</em>stractText>We anticipate that IV administration of C1-<em>INH</em> will significantly inhi<em>b</em>it complement mediated hemolysis in dogs with intravascular IMHA, as determined <em>b</em>y <em>b</em>lood <em>b</em>iomarker measurements (decreased plasma hemoglo<em>b</em>in, LDH and <em>b</em>iliru<em>b</em>in, increased haptoglo<em>b</em>in). We expect this will translate into significant reductions in transfusion requirements and duration of hospitalization.</A<em>b</em>stractText><A<em>b</em>stractText>This trial has <em>b</em>een prospectively registered with the AVMA registry (AAHSD005025).</A<em>b</em>stractText>

The potent hepatocarcinogen 3-methoxy-4-aminoazobenzene (3-MeO-AAB) has been reported to be bioactivated to mutagenic intermediates by rat liver microsomal cytochrome P450 (P450) and to be a selective inducer of rat P450IA2. In this study we have further investigated the roles of individual rat and human P450 enzymes in the bioactivation of this hepatocarcinogen in a Salmonella typhimurium TA1535/pSK1002 system where umu response is indicative of DNA damage. 3-MeO-AAB was found to be bioactivated by liver microsomal enzymes from rats and humans in this assay system. The liver microsomal activities are increased by pretreatment of rats with various P450 inducers such as phenobarbital (PB), beta-naphthoflavone (BNF), dexamethasone (DEX), acetone, ethanol, isoniazid (INH), diphenylhydantoin and valproic acid, and can be inhibited considerably by SKF-525A and metyrapone. alpha-Naphthoflavone (ANF) is also an inhibitor for the reaction catalyzed in BNF-treated rats, but stimulated the microsomal activity in DEX-treated rats. Evidence has also been obtained that specific antibodies raised against P450IIB1, P450IA1 or IA2, P450IIE1, and P450IIIA2 inhibited the activation in liver microsomes from rats pretreated with PB, BNF, INH and DEX respectively, suggesting the possible roles of several P450 enzymes in the bioactivation of 3-MeO-AAB. The results obtained with reconstituted monooxygenase systems containing various rat P450 enzymes are highly supportive of this conclusion. Human liver microsomal activation of 3-MeO-AAB was also inhibited to various extents by antibodies raised against P450IA2, P450MP, P450IIE1 and P450IIIA4. In a reconstituted system containing purified forms of human P450, P450IA2 was the most active in catalyzing 3-MeO-AAB, followed by P450IIIA4 and P450MP. ANF, a known activator of P450IIIA-catalyzed reactions, caused an increase in activation of 3-MeO-AAB in human liver microsomal and P450IIIA4- and P450MP-containing reconstituted systems. From these results it is concluded that multiple P450 enzymes in rat and human liver microsomes are involved in the bioactivation of 3-MeO-AAB, regardless of its selective induction of the rat P450IA2 gene.

Flaviviridae infections represent a major global health burden. By deciphering mechanistic aspects of hepatitis C virus (HCV)-host interactions, one could discover common strategy for inhibiting the replication of related flaviviruses. By elucidating the HCV interactome, we identified the 17-beta-hydroxysteroid dehydrogenase type 12 (HSD17B12) as a human hub of the very-long-chain fatty acid (VLCFA) synthesis pathway and core interactor. Here we show that HSD17B12 knockdown (KD) impairs HCV replication and reduces virion production. Mechanistically, depletion of HSD17B12 induces alterations in VLCFA-containing lipid species and a drastic reduction of lipid droplets (LDs) that play a critical role in virus assembly. Oleic acid supplementation rescues viral RNA replication and production of infectious particles in HSD17B12 depleted cells, supporting a specific role of VLCFA in HCV life cycle. Furthermore, the small-molecule HSD17B12 inhibitor, INH-12, significantly reduces replication and infectious particle production of HCV as well as dengue virus and Zika virus revealing a conserved requirement across Flaviviridae virus family. Overall, the data provide a strong rationale for the advanced evaluation of HSD17B12 inhibition as a promising broad-spectrum antiviral strategy for the treatment of Flaviviridae infections.

The role of extravascular lung water (EVLW) in the pathogenesis of inhalation injury (INH) when associated with concomitant major burn (B) remains controversial. Previous experimental models have investigated isolated INH without surface burn. This study measured the effects of isolated and combined INH on EVLW and pO2 in a porcine experimental model. The beneficial effects of early resuscitation with dextran-40 (DEX) were assessed, using a control group receiving standard Parkland formula (LR). In the first part of the study (INH vs. INH + B), a group of animals with a standardized INH was compared to a group also receiving a standardized 40% BSA third-degree surface burn (n = 8, each group). With serial measurements for 5 hours, EVLW was only modestly increased unless INH was accompanied by surface burn: 20.3 +/- 4.2 vs. 32.0 +/- 4.1 ml/kg at 5 hours (p less than 0.01). Similarly, pO2 fell much more dramatically in the INH + B group, 61 +/- 5 vs. 37 +/- 5 torr (p less than 0.05). The second part of the study compared standard Parkland crystalloid resuscitation with dextran-40 resuscitation in animals receiving a combined INH + B injury (LR vs. DEX, n = 8, each group). DEX resuscitation resulted in substantially lower accumulation of EVLW out to 5 hours, 34.1 +/- 5.0 vs. 13.1 +/- 3.0 ml/kg (p less than 0.01), and significantly better pO2, 35 +/- 5 vs. 64 +/- 4 torr (p less than 0.01).

CONCLUSIONS

Inhalation injury did not dramatically increase EVLW in this animal model unless accompanied by concomitant major surface burn. The deterioration in EVLW and pO2 seen in the combined injury was significantly improved with DEX resuscitation when compared to standard crystalloid resuscitation. Further study is indicated and clinical trials may be warranted.