High-throughput human primary cell-based airway model for evaluating influenza, coronavirus, or other respiratory viruses in vitro.

Influenza and other respiratory viruses present a significant threat to public health, national security, and the world economy, and can lead to the emergence of global pandemics such as from COVID-19. A barrier to the development of effective therapeutics is the absence of a robust and predictive preclinical model, with most studies relying on a combination of in vitro screening with immortalized cell lines and low-throughput animal models. Here, we integrate human primary airway epithelial cells into a custom-engineered 96-device platform (PREDICT96-ALI) in which tissues are cultured in an array of microchannel-based culture chambers at an air-liquid interface, in a configuration compatible with high resolution in-situ imaging and real-time sensing. We apply this platform to influenza A virus and coronavirus infections, evaluating viral infection kinetics and antiviral agent dosing across multiple strains and donor populations of human primary cells. Human coronaviruses HCoV-NL63 and SARS-CoV-2 enter host cells via ACE2 and utilize the protease TMPRSS2 for spike protein priming, and we confirm their expression, demonstrate infection across a range of multiplicities of infection, and evaluate the efficacy of camostat mesylate, a known inhibitor of HCoV-NL63 infection. This new capability can be used to address a major gap in the rapid assessment of therapeutic efficacy of small molecules and antiviral agents against influenza and other respiratory viruses including coronaviruses.

Infectivity of blood components from donors with occult hepatitis B infection - results from an Australian lookback programme.

BACKGROUND AND OBJECTIVES: Previous studies have demonstrated that transfused blood components from donors with occult hepatitis B virus infection (OBI) are potentially infectious. This study reports the results of an Australian lookback programme for the period subsequent to the commencement of individual donation HBV NAT in July 2010 and estimates the HBV transmission rate for components from two categories of donors, confirmed OBI and HBV inconclusive (anti-HBc reactive with non-discriminated NAT result). MATERIALS AND METHODS: Using the results of lookback investigations, we estimated HBV transmission rates by donor category and type of component transfused based on the prevalence of antibodies to HBV core antigen (anti-HBc) in recipients adjusted for the estimated prevalence in the general population. RESULTS: After subtracting the background anti-HBc rate, we derived an adjusted transmission rate (all components) with lower and upper bounds as follows: 0·85% (0·00-2·35%) for OBI donors, 2·83% (1·23-4·33%) for inconclusive donors and 1·81% (0·21-3·31%) for total (OBI and inconclusive) donors. The median adjusted transmission rate for total donors was higher (but not statistically) for plasma (3·01%) than RCCs (2·86%), but there was no evidence of transmission for cryoprecipitate or platelets (0% for both components). CONCLUSION: Our lookback study suggests a low (0·2-3·3%) but measurable rate of HBV transmission in Australia associated with donors with OBI and supports published evidence that at least some blood component types from OBI donors, including a proportion undetectable by ID-NAT can transmit HBV by transfusion.

Hyperplastic goitre and mortality in captive-reared black stilts ( Himantopus novaezelandiae ).

AIM: To study the gross, histopathological and clinico-pathological findings in cases of hyperplastic goitre in sub-adult captive- reared black stilts following their release on riverbeds in the south Canterbury region of New Zealand. METHODS: Necropsies were undertaken on the recovered carcasses of 48 black stilts over a 3-year period (1997-1999). The cause of death was determined, and thyroid glands were examined histopathologically and compared with those of free-living pied stilts. Concentrations of triiodothyronine (T3) and thyroxine (T4) in the serum of sub-adult and adult stilts were measured before and after iodine supplementation. RESULTS: The main causes of death of captive-reared black stilts following release were trauma, predation and starvation. An increase in size of the thyroid gland due to follicular hyperplasia and dilation was seen in all birds with intact thyroid glands (n=27). Dysplastic follicular changes such as epithelial desquamation, lipid deposition and haemorrhage were common in a large proportion of individuals with goitre. Dietary supplementation with iodine greatly improved survival rates in sub-adults following release, and significantly increased concentrations of T3 and T4 in serum. CONCLUSIONS: Subclinical goitre due to thyroid hyperplasia and dysplasia was the cause of hypothyroidism and this contributed to the poor survival of released sub-adult black stilts raised in captivity. Iodine supplementation of the diet of captive adults and sub-adults resulted in increased concentrations of T3 and T4 in serum and improved survivability.

The Charles River "hairless" rat mutation maps to chromosome 1: allelic with fuzzy and a likely orthologue of mouse frizzy.

Recent evidence has indicated that the recessive mutation affecting hypotrichosis in the Charles River (CR) "hairless" rat does not involve the hairless gene (hr) on rat chromosome 15. To determine if this mutation might be allelic (or orthologous) with any other previously mapped hypotrichosis-generating mutation in mammals, we have produced a panel of backcross rats segregating for the CR hairless rat mutation as well as numerous other markers from throughout the rat genome. Analysis of this panel has located the CR hairless rat's hypotrichosis-generating mutation on chromosome 1, near Myl2, where only the fuzzy mutation in rat (fz) and the frizzy mutation in mouse (fr) have been previously localized. Intercrossing fz/fz and CR hairless rats produced hybrid offspring with abnormal hair, showing that these two rat mutations are allelic. We suggest that the CR hairless rat mutation and fuzzy be renamed frizzy-Charles River (fr(CR)) and frizzy-Harlan (fr(H)), respectively, to reflect their likely orthology with the mouse fr mutation.

Video-microscopic analysis of dye coupling in the salamander olfactory bulb.

Cells in the mitral cell (MCL) and granule cell (GCL) layers of the olfactory bulb shape the representation of odor information in the brain. After intracellular Lucifer Yellow (LY) injections into lightly fixed olfactory bulb slices, clusters of dye coupled cells were previously observed in the MCL and GCL, but the relative extent of coupling in the two layers was unknown in adults. In the present study, the time course of LY coupling in the adult salamander olfactory bulb was quantified using video-microscopic methods. Analysis of fluorescent cell body counts showed that the incidence and the extent of LY coupling are greater in the GCL than in the MCL. With optimal low-current injection procedures, 97% of the injections into the GCL exhibited at least one coupled cell, and on average groups of six to eight cells were counted. Fewer injections into the MCL exhibited only one to three coupled cells. Some of these coupled cells were clearly mitral cells. No staining of cells was observed after extracellular LY injections, and intracellular injections of dextran dyes stained single cells, providing evidence that the LY coupled cells were stained through an intercellular route, presumably gap junctions. In live intact preparations, rapid LY staining of cell clusters was also observed using patch pipettes. Together, these results provide evidence that robust coupling occurs among olfactory bulb neurons in adults, which could have functional significance.

Effect of varying the optical zone diameter on the results of hyperopic laser in situ keratomileusis.

PURPOSE: To compare the predictability and safety of laser in situ keratomileusis (LASIK) for low to moderate spherical hyperopia using different ablation zone diameters. DESIGN: Retrospective, nonrandomized, comparative trial. PARTICIPANTS: Forty-nine eyes that underwent hyperopic LASIK. INTERVENTION: Two surgeons (JMD, RKM) performed hyperopic LASIK using the VISX STAR S2 excimer laser (VISX, Inc., Sunnyvale, CA) and the Bausch & Lomb Hansatome microkeratome (Chiron Vision, Irvine, CA) using ablation zone diameters of 5 x 9 mm, 5.5 x 8.5 mm, or 6 x 9 mm (the first number represents the optical zone diameter and the second number represents the diameter of the outer border of the ablation zone). MAIN OUTCOME MEASURES: Refractive and visual outcomes at 3 to 6 months after surgery were analyzed. Groups were compared for deviations from targeted spherical equivalent, uncorrected visual acuity, and loss of best spectacle-corrected visual acuity (BSCVA). RESULTS: The mean intended hyperopic correction was +2.48 +/- 1.13 diopters (D; 0.63-5.50 D). There were 16 eyes in the 5 x 9-mm group, 15 eyes in the 5.5 x 8.5-mm group, and 18 eyes in the 6 x 9-mm group. On average, the 5 x 9-mm group achieved 97% of the programmed correction, the 5.5 x 8.5-mm group achieved 104%, and the 6 x 9-mm group achieved 112% of the programmed correction. The tendency toward overcorrection in the 6 x 9-mm group compared with the 5 x 9-mm group was statistically significant (P < 0.05). The incidence of one line loss of BSCVA was greatest in the 5 x 9-mm group (19%) and lowest in the 6 x 9-mm group (6%). These differences were not statistically significant. No eyes experienced a loss of two or more lines of BSCVA at last examination. CONCLUSIONS: Hyperopic LASIK using the VISX STAR is safe and effective using different ablation zone diameters. There appears to be an increased tendency toward overcorrection with progressively larger optical zone diameters.

Symptoms of dry eye and recurrent erosion syndrome after refractive surgery.

PURPOSE: To determine the incidence and severity of patient complaints typical of dry eye and recurrent erosion syndrome after excimer laser refractive surgery and to compare the incidence of these symptoms after photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK). SETTING: Outpatient university practice. METHODS: A questionnaire was mailed to 1731 patients who had had primary myopic PRK or LASIK at least 6 months previously. Questions were designed to determine the incidence and character of ocular dryness and recurrent erosion symptoms and their impact on patient satisfaction and willingness to have surgery again. Responses from PRK and LASIK patients were compared. RESULTS: Responses from 231 PRK patients and 550 LASIK patients revealed an incidence of dryness symptoms in 43% and 48%, respectively (P >.05). Soreness of the eye to touch was reported by 26.8% and 6.7%, respectively (P <.0001). Sharp pains occurred in 20.4% of PRK patients and 8.0% of LASIK patients (P =.0001). Complaints of the eyelid sticking to the eyeball occurred in 14.7% and 5.6%, respectively (P =.0001). All symptoms occurred predominantly on waking. Frequency of eyelid sticking (P <.0005) and sharp pain (P <.005) symptoms, as well as severity of sharp pain symptoms (P <.0001), were significantly greater in PRK patients than in LASIK patients. On a scale of 0 to 10 (10 high), median overall patient satisfaction with surgery was 9 in both groups. Soreness of the eyelid to touch occurred significantly more frequently among patients with symptoms of sharp pains on waking (P <.001) and the sensation of the eyelid sticking to the eyeball (P <.001). Patients with 1 or more symptoms were twice as likely as asymptomatic patients to have a satisfaction score of less than 8 (P <.001). CONCLUSIONS: Ocular dryness symptoms occurred commonly after PRK and LASIK. Symptoms suggestive of mild recurrent erosions included sharp pains, the sensation of the eyelid sticking to the eyeball, and soreness of the eyelid to touch, a previously unrecognized symptom of this condition. These symptoms occurred commonly after excimer laser procedures but were significantly more common, more severe, and more prolonged after PRK. The presence of these symptoms had a significant effect on patient satisfaction.

Conductive keratoplasty for the correction of hyperopia.

BACKGROUND/PURPOSE: Conductive keratoplasty (CK) is a surgical technique that delivers radio frequency (350 kHz) current directly into the corneal stroma through a Keratoplasty tip inserted into the peripheral cornea at 8 to 32 treatment points. A full circle of CK spots produces a cinching effect that increases the curvature of the central cornea, thereby decreasing hyperopia. We report here the 12-month results of a 2-year, prospective, multicenter US clinical trial conducted to evaluate the efficacy, safety, and stability of CK. METHODS: A total of 233 patients (401 eyes) with preoperative hyperopia of +0.75 to +3.00 D and < or = 0.75 D of astigmatism (mean preoperative manifest refractive spherical equivalent = +1.76 D +/- 0.60) were enrolled into the study at 13 centers and underwent CK treatment. RESULTS: Twelve-month postoperative data are available on 203 eyes for safety and stability and 171 eyes for safety, stability, and efficacy. A total of 91% had uncorrected visual acuity (UCVA) of 20/40 or better, and 51% had UCVA of 20/20 or better. Manifest refractive spherical equivalent was within +/- 0.50 D in 58%, within +/- 1.00 D in 91%, and within +/- 2.00 D in 99%. The mean change in residual refraction was 0.26 D +/- 0.49 between 3 and 6 months, 0.09 D +/- 0.37 between 6 and 9 months, and 0.13 D +/- 0.39 between 9 and 12 months. CONCLUSIONS: One-year data show safety and efficacy of CK in the treatment of hyperopia. Changes in residual refractive error after CK appeared to be small, suggesting that a stable refraction could be achieved by 6 months.

Postoperative inflammation, microbial complications, and wound healing following laser in situ keratomileusis.

Although the biology of corneal wound healing is only partly understood, healing after photorefractive keratectomy (PRK) and laser in situ keratomileusis (LASIK) differs in many respects, and the mechanisms appear to be differently controlled. There is less of an inflammatory and healing response after LASIK, but a longer period of sensory denervation. The cellular, molecular, and neural regulatory phenomena associated with postoperative inflammation and wound healing are likely to be involved in the adverse effects after LASIK, such as flap melt, epithelial ingrowth, and regression. Interface opacities in the early postoperative period include diffuse lamellar keratitis (DLK), microbial keratitis, epithelial cells, and interface opacities. Diffuse lamellar keratitis (sands of the Sahara syndrome) describes an apparently noninfectious diffuse interface inflammation after lamellar corneal surgery probably caused by an allergic or a toxic inflammatory reaction. Noninfectious keratitis must be distinguished from microbial keratitis to avoid aggressive management and treatment with antimicrobial drugs. Microbial keratitis is a serious complication after LASIK, but a good visual outcome can be achieved following prompt and appropriate treatment.

Epithelial ingrowth after laser in situ keratomileusis.

PURPOSE: To report the incidence and risk factors for clinically significant epithelial ingrowth after laser in situ keratomileusis as well as the recurrence rate and visual outcomes after its treatment. METHODS: We defined clinically significant epithelial ingrowth as that which required surgical removal. From a cohort of 3, 786 eyes that underwent primary laser in situ keratomileusis from February 1996 to August 1998 and its derivative of 480 eyes that later underwent enhancement laser in situ keratomileusis by one surgeon (R.K.M.), we identified all eyes with clinically significant epithelial ingrowth. RESULTS: The incidence of significant epithelial ingrowth after primary treatment was 35 (0.92%) of 3,786 eyes. The incidence after enhancement treatment was eight (1.7%) of 480 eyes (p = NS). Fourteen of 43 eyes had a postoperative epithelial defect associated with subsequent development of epithelial ingrowth. Six of 43 eyes had loose epithelium intraoperatively, suggesting epithelial basement membrane dystrophy. Epithelial ingrowth was treated by lifting the flap, scraping the bed and the posterior surface of the flap, and replacing the flap without the use of caustic agents. In 42 of 43 eyes, the epithelial ingrowth under the flap was continuous with the surface epithelium. Clinically significant ingrowth recurred in 10 of 43 eyes after the initial surgical removal. CONCLUSIONS: Clinically significant epithelial ingrowth is an infrequent complication of laser in situ keratomileusis. We hypothesize that epithelial ingrowth is secondary to postoperative invasion under the flap by surface epithelial cells rather than intraoperative implantation of epithelial cells. Treatment should consist of complete mechanical removal of epithelium from the posterior surface of the corneal flap and keratectomy bed and ensuring tight apposition of the flap with the bed.

Quantitative topographic irregularity as a predictor of spectacle-corrected visual acuity after refractive surgery.

PURPOSE: To evaluate a new topographic index called topographic irregularity as a quantitative predictor of corrected vision after refractive surgery. METHODS: We defined topographic irregularity as the summed difference at all points between a topographic refractive corneal power map and its best-fit spherocylinder. We prospectively studied 107 eyes of 107 patients 3 months after a variety of refractive procedures. Topographic irregularity was calculated from topographic maps, and the correlation between topographic irregularity and spectacle-corrected visual acuity was determined using both high-contrast and low-contrast acuity charts. This correlation was compared with correlations for the surface regularity index and the surface asymmetry index. Next, we analyzed 54 of these topographic maps to create a regression scale relating surface regularity index, surface asymmetry index, and topographic irregularity to predict spectacle-corrected visual acuity. This scale was then used to predict spectacle-corrected visual acuity on the remaining 53 postoperative patients. RESULTS: The correlation of topographic irregularity with spectacle-corrected visual acuity (R(2) =.36) was comparable to the correlation for the surface regularity index (R(2) =.36) and stronger than for the surface asymmetry index (R(2) =.11) when spectacle-corrected visual acuity was measured with high-contrast eye charts. Topographic irregularity correlated more strongly with spectacle-corrected visual acuity (R(2) =.42) than either the surface regularity index (R(2) =.28) or the surface asymmetry index (R(2) =.14) when spectacle-corrected visual acuity was measured with low-contrast eye charts. Using the regression scale, prediction of high-contrast and low-contrast spectacle-corrected visual acuity from topographic irregularity was superior to or comparable to predictions using the surface regularity index and the surface asymmetry index. CONCLUSIONS: Topographic irregularity has a closer correlation with spectacle-corrected visual acuity than existing topographic indexes. Topographic irregularity is also an accurate predictor of spectacle-corrected visual acuity and may be a more sensitive tool for evaluating postoperative visual performance than current topographic measures.

Cardiac excitation-contraction coupling in the portal hypertensive rat.

Basal contractility and responses to beta-adrenoceptor activation are compromised in hearts from rats with chronic portal vein stenosis. Here we report the effect of partial ligation of the portal vein on myocardial G protein expression, beta-adrenoceptor-G protein coupling, and excitation-contraction coupling (ECC). Contractility (dT/dt) was reduced 30-50% in right and left ventricles, but the rate of relaxation (-dT/dt) was unaffected. Isoproterenol-induced positive inotropism was diminished, but there was no difference in ED(50). The concentration-dependent increase in -dT/dt was unaffected. G(s)alpha and G(i)alpha expression, cholera toxin- and pertussis toxin-induced ADP-ribosylation, and formation of the agonist-receptor-G(s) complex were unaffected by portal vein stenosis. Of the components of ECC examined, the caffeine-sensitive sarcoplasmic reticulum Ca(2+) pool was reduced 35%, although the Ca(2+) uptake and release processes were unchanged; the apparent density of L-type Ca(2+) channels decreased 60% with no change in affinity; the dihydropyridine Ca(2+) channel agonist BAY K 8644 produced relative changes in dT/dt that were similar in both groups, suggesting normal function in the remaining Ca(2+) channels; and Na(+)/Ca(2+) exchange was reduced 50% in the portal vein stenosis group. These data suggest that the effect of portal vein stenosis on the myocardium is the result of alterations to ECC.

Microkeratome complications of laser in situ keratomileusis.

PURPOSE: To evaluate the incidence, types, and outcome of microkeratome complications that occur during laser in situ keratomileusis (LASIK). DESIGN: Retrospective, noncomparative, case series. PARTICIPANTS: Three thousand nine hundred ninety-eight eyes that underwent primary LASIK by four surgeons between November 1996 and August 1998 at a university-based refractive center. METHODS: All cases with significant microkeratome complications leading to abandonment of the LASIK procedure were identified and reviewed. MAIN OUTCOME MEASURES: Incidence of complications, change in best corrected visual acuity (BCVA), change in refractive error, and types of complication. RESULTS: There were 27 complications leading to abandonment of the LASIK procedure of 3998 eyes. The overall rate of microkeratome complication was 1 in 150 (0.68%), but it was 1 in 77 (1.3%) in the surgeons' first 1000 eyes, decreasing to 1 in 250 (0.4%) in the last 1000 eyes. Of the 24 planned bilateral cases, 15 complications (63%) happened on the first operated eye. Twenty-six of 27 eyes (96%) recovered to within one line of preoperative BCVA, and one eye lost two lines. At last examination before any repeat refractive procedures, spherical equivalent manifest refraction returned to within 1 diopter (D) of its preoperative value in 18 of 19 eyes (95%), and astigmatism in 16 of 19 eyes (84%) returned to within 1 D of its preoperative value. Sixteen of 27 eyes (59%) had repeat LASIK. Two eyes had complications at repeat LASIK, one of which led to abandonment of the LASIK procedure for a second time. CONCLUSIONS: There is a significant learning curve in the use of the microkeratome. If ablation is not performed, flap complications rarely lead to significant visual loss and generally do not result in a change in refractive error.

Deep lamellar keratoplasty using viscoelastic dissection.

We describe a technique for performing deep lamellar keratoplasty using viscoelastic dissection. Deep lamellar dissections of the cornea using viscoelastic substances (sodium hyaluronate) were performed on 4 eyes of 4 patients. One patient with keratoconus and another with corneal scarring underwent lamellar keratoplasty using the technique as the sole procedure for visual rehabilitation. Two patients (2 eyes) with opaque corneas underwent deep lamellar dissection with removal of stromal tissue to allow visualization of the anterior segment structures prior to penetrating keratoplasty, thereby facilitating separation of iridocorneal adhesions as the Descemet membrane was incised. Deep lamellar dissection was performed without complications related to the procedure in all 4 eyes. The 2 lamellar grafts cleared completely, and both eyes achieved excellent visual acuity with spectacle correction. In the other 2 eyes, deep lamellar dissection provided clear visualization of anterior segment structures during incision of the Descemet membrane. Deep lamellar dissection using viscoelastic substances is a useful technique during lamellar keratoplasty.