Maternal varicella antibodies in children aged less than one year: Assessment of antibody decay.

OBJECTIVES: To investigate maternal antibody levels to varicella in infants <12 months of age in Ontario, Canada. STUDY DESIGN: In this study, we included specimens from infants <12 months of age, born at ≥37 weeks gestational age, who had sera collected at The Hospital for Sick Children (Toronto, Canada) between 2014-2016. We tested sera using a glycoprotein-based enzyme-linked immunosorbent assay (gpELISA). We measured varicella susceptibility (antibody concentration <150mIU/mL) and mean varicella antibody concentration, and assessed the probability of susceptibility and concentration between one and 11 months of age using multivariable logistic regression and Poisson regression. RESULTS: We found that 32% of 196 included specimens represented infants susceptible to varicella at one month of age, increasing to nearly 80% at three months of age. At six months of age, all infants were susceptible to varicella and the predicted mean varicella antibody concentration declined to 62 mIU/mL (95% confidence interval 40, 84), well below the threshold of protection. CONCLUSIONS: We found that varicella maternal antibody levels wane rapidly in infants, leaving most infants susceptible by four months of age. Our findings have implications for the timing of first dose of varicella-containing vaccine, infection control measures, and infant post-exposure prophylaxis recommendations.

Uptake of pertussis immunization in pregnancy and determinants of vaccination in Toronto, Canada.

INTRODUCTION: Pertussis causes significant morbidity and mortality in infants aged <6 months. Maternal pertussis vaccination during pregnancy has been recommended in Canada since 2018 to reduce these negative outcomes. In the absence of routine immunization coverage data, our objective was to evaluate uptake in Toronto, Canada. METHODS: We recruited mother-infant pairs at The Hospital for Sick Children, Toronto, between 2018 and 2020. We performed logistic regression to examine associations between demographics and self-reported pertussis vaccination. RESULTS: 76/243 mothers (31.3 %) reported receiving pertussis vaccination during their most recent pregnancy. Odds of receiving vaccination more than doubled with each 1-year increase in year of pregnancy (aOR: 2.2; 95 % CI: 1.3, 3.6; p < 0.01) and among those born in Canada as compared to those not (aOR: 2.0; 95 % CI: 1.1, 3.6; p = 0.02) CONCLUSION: Uptake of pertussis vaccination during pregnancy in Ontario has increased in recent years, however coverage remains lower than desirable.

Epidemiology of cytomegalovirus antiviral resistance testing for solid organ and bone marrow transplant patients from 2011 - 2019.

BACKGROUND: CMV reactivation post-transplantation is common, with need for prompt identification of patients most at-risk for CMV antiviral drug resistance (AVDR). OBJECTIVES: This study describes CMV AVDR frequencies, antiviral prescribing practices, and AVDR risk factors in patients from 2011 to 2019 in British Columbia, Canada. STUDY DESIGN: Retrospective review of demographics, transplant type, viral loads, antiviral exposure duration, and 12-month mortality was conducted for all patients with samples submitted for CMV AVDR testing from 2011 to 2019. Genotyping of AVDR mutations occurred at the national reference laboratory. Mann-Whitney U, T-test or Fisher's exact tests examined differences between patients with and without AVDR. RESULTS: Fifty-three plasma and three tissue/fluid specimens successfully underwent CMV AVDR testing; of these samples, 27/56 (48%) had AVDR mutations detected. The commonest AVDR mutations were at UL97 loci A594 (20%), H596 (12%) and L595 (12%). Mutations occurred more frequently in requests from solid organ than hematopoietic stem cell transplant patients (58% vs. 27%, p = 0.05). Previous resistance testing was a significant risk factor for AVDR (p < 0.001). Patients with AVDR had approximately 51 more days of antiviral therapy (p = 0.007) and took 9 days longer to clear viremia (p = 0.23). The median turnaround time from sample send-out to reporting was nine days. However, empiric use of second-line antivirals occurred in most cases (39/53, 74%) before results were available. DISCUSSION: Laboratories should strive to provide timely CMV AVDR testing for transplant patients, to minimize unnecessary exposure to second-line antiviral agents. The findings of this study may help guide clinicians when selecting empiric antiviral therapy.

Population immunity to measles in Canada using Canadian Health Measures survey data - A Canadian Immunization Research Network (CIRN) study.

We aimed to determine population immunity to measles in Canada, and to assess the risk of future outbreaks. We tested 11,176 sera from Cycles 2 (2009-2011) and 3 (2011-2013) cohorts from the biobank of Statistics Canada's Canadian Health Measures Survey (CHMS) using the BioPlex 2220 MMRV IgG assay. We then tested all BioPlex negative and equivocal samples using a more sensitive Plaque Reduction Neutralization Test (PRNT). We determined the weighted proportion of positive, equivocal, and negative samples by age, sex, region and whether individuals were born in Canada. We found that 90.0% (95% confidence interval (CI): 88.2, 91.9) of samples were positive, 4.5% (95% CI: 3.4, 5.5) were equivocal and 5.5% (95% CI: 4.3, 6.7) were negative. Individuals in the 12-19 year age band had the lowest proportion positive at 78.7% (95% CI: 74.2, 83.2) and the highest proportion of positive samples was found in those 60-79 years (99.6%, 95% CI: 99.3, 99.9). Seropositivity was consistently <90% across a broad range of pediatric and adult age bands (6-39 years). We found that a slightly higher proportion of females were positive (91.9%, 95% CI: 90.1, 93.6) compared to males (88.3%, 95% CI: 85.8, 90.7). When taking into account interaction between age and born in Canada status, we found individuals born in Canada aged 19 and under were less susceptible (OR = 0.6 (95% CI: 0.4, 0.95)) compared to those born outside Canada whereas, those aged 20 and over were more susceptible (OR = 1.7 (95% CI: 1.1, 2.8)). Our findings indicate that measles immunity in Canada is below the 95% immunity threshold required to sustain measles elimination, underscoring the importance of maintaining high vaccine coverage to prevent future measles outbreaks and sustain Canada's elimination status.

Optimisation of methodology for whole genome sequencing of Measles Virus directly from patient specimens.

In an era of decreasing genetic diversity of Measles Virus (MeV), effective surveillance requires a higher-resolution genotyping method or whole genome sequencing (WGS) to document elimination. Through optimization of MeV WGS protocol, we developed a MeV-specific probe enrichment method that allows next generation sequencing from clinical specimens. With the probe enrichment method, 70% of specimens can be sequenced at a read depth of greater than 10 reads with minimal off-target sequences.

Next-generation sequencing for cytomegalovirus antiviral resistance genotyping in a clinical virology laboratory.

INTRODUCTION: The identification of CMV antiviral drug resistance (AVDR) is a critical diagnostic test for immunocompromised patients with CMV infection and a failure of virologic response on optimal antiviral treatment. We developed a next-generation sequencing (NGS) assay for CMV AVDR testing and compared the AVDR mutations identified by NGS to Sanger sequencing. METHODS: Retrospective review of CMV AVDR testing requests for UL97 and UL54 at our laboratory from 2014 to 2019 was conducted. NGS was performed on the MinION and compared to Sanger sequencing performed at the national reference laboratory. Analysis of the sequences was completed with a novel cloud bioinformatics platform (BugSeq). RESULTS: Twenty patient samples previously characterized were included for study on the MinION. NGS captured all of the CMV AVDR mutations identified by Sanger, and identified additional mutations in UL97 and/or UL54 in 8/13 (62%) of the samples. An analysis of the depth of coverage at which we no longer detected minority single nucleotide variants (SNVs) detected in the original data was conducted, estimating a recall of 95% at 1800 fold coverage. CONCLUSION: NGS utilizing MinION technology for the detection of CMV AVDR mutations identified additional minority variants in UL97 and UL54 as compared with Sanger sequencing. Through the application of a bioinformatics pipeline available online, our NGS process eliminates barriers associated with the use of the MinION and NGS in clinical laboratories.

An enrichment method for capturing mumps virus whole genome sequences directly from clinical specimens.

Recently there has been a significant increase in the number of mumps outbreaks occurring in highly vaccinated populations. These outbreaks are often due to a mumps genotype G virus, where sequencing of the SH gene does not reveal enough genetic diversity to sufficient to resolve outbreaks. This has elevated the need to be able to sequence complete mumps viruses from clinical samples without laborious methods. Here we describe a probe enrichment method that allows for whole genome sequencing of the mumps virus directly from clinical specimens. Using 136 clinical samples, we show this method allows for a significant increase in the percentage of viral sequencing reads, resulting in the capture of mumps genomes. This method will be an asset in investigating future mumps outbreaks.

Evaluation of a commercially-available surrogate virus neutralization test for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2).

There remains an urgent need for assays to quantify humoral protective immunity to SARS-CoV-2 to understand the immune responses of COVID-19 patients, evaluate efficacy of vaccine candidates in clinical trials, and conduct large-scale epidemiological studies. The plaque-reduction neutralization test (PRNT) is the reference-standard for quantifying antibodies capable of neutralizing SARS-CoV-2. However, the PRNT is logistically demanding, time-consuming, and requires containment level-3 facilities to safely work with live virus. In contrast, a surrogate virus neutralization test (sVNT) manufactured by Genscript is a quick and simple assay that detects antibodies that inhibit the RBD-ACE2 interaction, crucial for virus entry into host cells. In this study, we evaluate the sensitivity, specificity, and cross-reactivity of the sVNT compared with the PRNT using both 50% and 90% SARS-CoV-2 neutralization as a reference-standard. We found that the sVNT provides a high-throughput screening tool prior to confirmatory PRNT testing for the evaluation of SARS-CoV-2 neutralizing antibodies.

Measles Antibody Levels in Young Infants.

BACKGROUND: Infants are often assumed to be immune to measles through maternal antibodies transferred during pregnancy and, in many countries, receive their first measles-containing vaccine at 12 to 15 months. Immunity may wane before this time in measles-eliminated settings, placing infants at risk for measles and complications. We investigated humoral immunity to measles in infants <12 months of age in Ontario, Canada. METHODS: We selected sera collected at a tertiary pediatric hospital from infants <12 months who were born at ≥37 weeks' gestational age. We excluded infants with conditions that affect antibody levels. We selected ≤25 sera from 8 predetermined age bands and tested them for measles-neutralizing antibody using the plaque-reduction neutralization test. We calculated the proportion immune at each age band, and predictors of infant susceptibility were assessed by using multivariable logistic regression and Poisson regression. RESULTS: Of 196 infant sera, 56% (110 of 196) were from boys, and 35% (69 of 196) were from infants with underlying medical conditions. In the first month, 20% (5 of 25) of infants had antibodies below the protective threshold, which increased to 92% (22 of 24) by 3 months. By 6 months, all infants had titers below the protective threshold. In a multivariable analysis, infant age was the strongest predictor of susceptibility (odds ratio = 2.13 for each additional month increase; 95% confidence interval: 1.52-2.97). CONCLUSIONS: Most infants were susceptible to measles by 3 months of age in this elimination setting. Our findings inform important policy discussions relating to the timing of the first dose of measles-containing vaccine and infant postexposure prophylaxis recommendations.

Calibration and Evaluation of Quantitative Antibody Titers for Varicella-Zoster Virus by Use of the BioPlex 2200.

Most commercially available enzyme immunoassay-based methods have limited sensitivity to detect antibody responses to varicella-zoster virus (VZV) in vaccinated individuals, who produce lower antibody levels than those with natural infection. However, more sensitive methods are either not commercially available or less amenable to high-throughput testing. The BioPlex 2200 measles, mumps, rubella, and varicella (MMRV) IgG assay (Bio-Rad Laboratories, Hercules, CA) is an automated high-throughput platform based on the microsphere Luminex technology that measures antibodies against measles, mumps, rubella, and varicella viruses simultaneously. Although it has U.S. Food and Drug Administration approval as a qualitative diagnostic test for measles, mumps, rubella, and varicella virus immunity, in this study, we have validated the assay to produce quantitative titers (off label) against the VaccZyme VZV glycoprotein (VZVgp) low-level IgG kit (The Binding Site Ltd., Birmingham, UK) using the World Health Organization international standard. Here, we show that the BioPlex 2200 MMRV IgG assay has sensitivity superior to that of the Zeus enzyme-linked immunosorbent assay (ELISA) VZV IgG assay (Zeus Diagnostics, Branchburg, NJ). Using receiver operating characteristic (ROC) analysis and adjusting the cutoff levels, we improved the sensitivity of the quantitative BioPlex 2200 MMRV IgG assay to 97.4%, while maintaining 100% specificity.

Assessment of population immunity to measles in Ontario, Canada: a Canadian Immunization Research Network (CIRN) study.

Canada eliminated measles in 1998. We conducted a sero-epidemiology study to estimate population immunity to measles in the province of Ontario, Canada and to identify groups at higher risk of outbreaks. We used a previously developed modified enzyme immunoassay to test 1,199 residual sera from patients aged 1-39 years. We re-tested negative and equivocal sera using a plaque reduction neutralization assay. We interpreted our results in the context of Ontario's immunization program and vaccine coverage data. Of 1,199 sera, 1035 (86.3%, 95% confidence interval (CI) 84.4, 88.2) were above the measles threshold for protection, 70 (5.8%, 95% CI 4.5, 7.2) were equivocal and 94 (7.8%, 95% CI 6.3, 9.4) were negative. The proportion of positive sera was highest for those 1-5 years, with 180/199 (90.5%, 95% CI 86.4, 94.5) positive sera, and lowest for those age 12-19 years, at 158/199 (79.4%, 95% CI 73.8, 85.0). Adjusted for age, females were more likely than males to have antibody titers above the threshold of protection (odds ratio = 1.60, 95% CI 1.14, 2.24). Most of the study cohort were eligible for two measles vaccine doses, and vaccine uptake in Ontario is >90% for school-aged cohorts. We observed a higher than expected proportion of sera with antibody levels below the threshold of protection, suggesting that immunity in some Ontario age-groups may be waning, despite high vaccine coverage. Alternatively, the traditional measles correlates of protection may not be an appropriate measure of population protection in measles-eliminated settings.

Calibration and Evaluation of Quantitative Antibody Titers for Measles Virus by Using the BioPlex 2200.

The BioPlex 2200 (Bio-Rad Laboratories, Hercules, CA) is a rapid, automated platform, which can screen large numbers of specimens for antibodies to measles, mumps, rubella, and varicella. Although approved for producing qualitative results, in this study we validated the test (off-label) to allow reporting of quantitative results. To do this, we used the third anti-measles World Health Organization standard to generate a calibration curve that allowed relative fluorescence intensity to be translated into quantitative antibody titer (antibody units [AU]/ml). The results from the BioPlex 2200 and the reference plaque reduction neutralization test (PRNT) exhibited a reasonable correlation following an exponential function, but correlation was poor in low-titer samples. Using a receiver operating characteristics analysis, an equivocal zone for the BioPlex 2200 was established between ≥0.13 and <1.10 AU/ml to achieve 100% specificity (95% confidence interval [CI] = 83.2 to 100%) and 100% sensitivity (95% CI = 93.5 to 100%) versus PRNT. By determining an equivocal range requiring confirmation by PRNT, we can avoid underestimating the levels of immunity through false-negative results and optimize methods for seroepidemiological studies.

ODDD-linked Cx43 mutants reduce endogenous Cx43 expression and function in osteoblasts and inhibit late stage differentiation.

INTRODUCTION: Bone development and modeling requires precise gap junctional intercellular communication (GJIC). Oculodentodigital dysplasia (ODDD) is an autosomal dominant human disease caused by mutations in the gene (GJA1) encoding the gap junction protein, connexin43 (Cx43). The disease is characterized by craniofacial bone deformities and limb abnormalities. It is our hypothesis that Cx43 mutation causes osteoblast dysfunction, which may contribute to the bone phenotype of ODDD. MATERIALS AND METHODS: We expressed human and mouse ODDD-linked Cx43 mutants in MC3T3-E1 cells and primary mouse osteoblasts by retroviral infection and evaluated their in vitro differentiation as an index of osteoblast function. We compared these findings to the differentiation of osteoblasts isolated from a mouse model of ODDD that harbors a germ line Cx43 mutation and exhibits craniofacial and limb defects mimicking human ODDD. We determined the differentiation status of osteoblasts by analyzing alkaline phosphatase activity and the expression levels of osteoblast markers including bone sialoprotein and osteocalcin. RESULTS: We showed that ODDD-linked Cx43 mutants are loss-of-function and dominant-negative to co-expressed Cx43 and, furthermore, greatly inhibit functional GJIC in osteoblasts. Surprisingly, the mutants had only a minor effect on osteoblast differentiation when introduced into lineage committed cells. In contrast, osteoblasts isolated from the ODDD mouse model exhibited impaired late stage differentiation. CONCLUSIONS: Expression of human and mouse ODDD-linked Cx43 mutants failed to significantly impair differentiation in cells predisposed to the osteoblast lineage; however, germ line reduction of Cx43-based GJIC leads to impaired osteoblast differentiation, which may account for the bone phenotypes observed in ODDD patients.

Connexins and gap junctions in mammary gland development and breast cancer progression.

The development and function of the mammary gland require precise control of gap junctional intercellular communication (GJIC). Here, we review the expression and function of gap junction proteins, connexins, in the normal mouse and human mammary gland. We then discuss the possible tumor-suppressive role of Cx26 and Cx43 in primary breast tumors and through the various stages of breast cancer metastasis and consider whether connexins or GJIC may actually promote tumorigenesis at some stages. Finally, we present in vitro data on the impact of connexin expression on breast cancer cell metastasis to the bone. We observed that Cx43 expression inhibited the invasive and migratory potentials of MDA-MB-231 breast cancer cells in a bone microenvironment, provided by the MC3T3-E1 mouse osteoblastic cell line. Expression of either Cx26 or Cx43 had no effect on MDA-MB-231 growth and adhesion under the influence of osteoblasts and did not result in regulation of osteogenic gene expression in these breast cancer cells. Furthermore, connexin-expressing MDA-MB-231 cells did not have an effect on the growth or differentiation of MC3T3-E1 cells. In summary, we conclude that connexin expression and GJIC are integral to the development and differentiation of the mammary gland. In breast cancer, connexins generally act as tumor suppressors in the primary tumor; however, in advanced breast tumors, connexins appear to act as both context-dependent tumor suppressors and facilitators of disease progression.

Connexins act as tumor suppressors in three-dimensional mammary cell organoids by regulating differentiation and angiogenesis.

Connexins are tumor suppressors, and human breast connexin 26 (Cx26) and connexin 43 (Cx43) gap junctions are often down-regulated in breast cancer. We previously showed that Cx26 and Cx43 overexpressed in MDA-MB-231 breast cancer cells inhibited tumor growth in vivo but not in two-dimensional cultures. In the current study, we show that overexpression of Cx26 or Cx43 has tumor-suppressive properties in a three-dimensional environment such that they reduced anchorage-independent cell growth and induced partial redifferentiation of three-dimensional organoids of MDA-MB-231 cells. Importantly, the majority of exogenous connexins did not localize to the cell-cell interface or rescue gap junctional intercellular communication (GJIC) as assessed by dye transfer, providing evidence of a GJIC-independent mechanism of mammary tumor suppression. To further elucidate the mechanisms involved in connexin-induced three-dimensional redifferentiation of tumor cells, we examined whether connexin expression has a role in epithelial to mesenchymal transition (EMT). Cx26 and Cx43 reduced cell migration, increased cytokeratin 18 expression, and decreased vimentin levels, indicating a shift from a mesenchymal towards an epithelial phenotype. In addition, we examined the role of connexins in angiogenesis by probing an angiogenesis antibody array with conditioned media from three-dimensional MDA-MB-231 cultures. This revealed that connexin overexpression regulated various angiogenesis-linked proteins. Furthermore, secreted factors from connexin overexpressing cells inhibited endothelial cell tubulogenesis and migration, and xenografts of Cx43 overexpressing MDA-MB-231 cells showed reduced tumor angiogenesis. In summary, Cx26 and Cx43 inhibit the malignant properties of MDA-MB-231 cells via GJIC-independent mechanisms, including regulation of EMT and angiogenesis.

Down-regulation of Cx43 by retroviral delivery of small interfering RNA promotes an aggressive breast cancer cell phenotype.

Connexins are gap junction proteins that assemble into channels that mediate direct intercellular communication. Connexins are well-documented tumor suppressors and are thought to regulate both cell growth and differentiation. As previously reported, most human breast tumors and cell lines down-regulate gap junctions or have defective gap junctional intercellular communication. Furthermore, overexpression of connexins in breast cancer cells inhibits tumor growth in vivo. In this study, we hypothesize that controlled Cx43 down-regulation would induce breast tumor cells to acquire a more aggressive phenotype. Here we report that Cx43 was down-regulated in both normal rat kidney (NRK) cells and human breast cancer cell lines (MDA-MB-231 and Hs578T) by transfection with chemically synthesized small interfering RNA (siRNA) or short hairpin RNA generated from a retroviral infection. Furthermore, we show that retroviral delivery and expression of siRNA directed to different coding regions of Cx43 resulted in differential levels of Cx43 silencing and impaired gap junctional intercellular communication. Cx43-silenced Hs578T cells grew faster and were more migratory. Finally, Western blot analysis revealed that down-regulation of Cx43 resulted in decreased expression of thrombospondin-1, an antiangiogenesis molecule, and increased expression of vascular endothelial growth factor. Taken together, these results suggest that Cx43 is required for maintaining cell differentiation and the regulation of molecules important in angiogenesis.

Oculodentodigital dysplasia-causing connexin43 mutants are non-functional and exhibit dominant effects on wild-type connexin43.

Oculodentodigital dysplasia, a rare condition displaying congenital craniofacial deformities and limb abnormalities, has been associated with over 20 known human connexin43 (Cx43) mutations. The localization of two of these mutants, G21R and G138R, was examined in Cx43-positive normal rat kidney cells (NRK) and Cx43-negative gap junctional intercellular communication-deficient HeLa cells. Green fluorescent protein-tagged and untagged Cx43 G21R and G138R mutants were transported to the plasma membrane and formed punctate structures reminiscent of gap junction plaques in both NRK and HeLa cells. Further localization studies revealed no significant trafficking defects as subpopulations of Cx43 mutants were found in both the Golgi apparatus and lysosomes, not unlike wild-type Cx43. Dual patch clamp functional analysis of the mutants expressed in gap junctional intercellular communication-deficient N2A cells revealed that neither G21R nor G138R formed functional gap junction channels, although they successfully reached cell-cell interfaces between cell pairs. Importantly, when either mutant was expressed in NRK cells, dye coupling experiments revealed that both mutants inhibited endogenous Cx43 function. These studies suggest that, although patients suffering from oculodentodigital dysplasia possess one wild-type Cx43 allele, it is likely that Cx43-mediated gap junctional intercellular communication is reduced below 50% because of a dominant-negative effect of mutant Cx43 on wild-type Cx43.

Functional characterization of oculodentodigital dysplasia-associated Cx43 mutants.

Oculodentodigital dysplasia (ODDD) is associated with at least 28 connexin43 (Cx43) mutations. We characterized four of these mutants; Q49K, L90V, R202H, and V216L. Populations of these GFP-tagged mutants were transported to the cell surface in Cx43-negative HeLa cells and Cx43-positive NRK cells. Dual patch-clamp functional analysis in N2A cells demonstrated that channels formed by each mutant have dramatically reduced conductance. Dye-coupling analysis revealed that each mutant exhibits a dominant-negative effect on wild-type Cx43. Since ODDD patients display skeletal abnormalities, we examined the effect of three other Cx43 mutants previously shown to exert dominant-negative effects on wild-type Cx43 (G21R, G138R, and G60S) in neonatal calvarial osteoblasts. Differentiation was unaltered by expression of these mutants as alkaline phosphatase activity and extent of culture mineralization were unchanged. This suggests that loss-of-function Cx43 mutants are insufficient to deter committed osteoblasts from their normal function in vitro. Thus, we hypothesize that the bone phenotype of ODDD patients may result from disrupted gap junctional intercellular communication earlier in development or during bone remodeling.

Biophysical characterization of zebrafish connexin35 hemichannels.

A subset of connexins can form unopposed hemichannels in expression systems, providing an opportunity for comparison of hemichannel gating properties with those of intact gap junction channels. Zebrafish connexin35 (Cx35) is a member of the Cx35/Cx36 subgroup of connexins highly expressed in the retina and brain. In the present study, we have shown that Cx35 expression in Xenopus oocytes and N2A cells produced large outward whole cell currents on cell depolarization. Using whole cell, cell-attached, and excised patch configurations, we obtained multichannel and single-channel current recordings attributable to the Cx35 hemichannels (I(hc)) that were activated and increased by stepwise depolarization of membrane potential (V(m)) and deactivated by hyperpolarization. The currents were not detected in untransfected N2A cells or in control oocytes injected with antisense Cx38. However, water-injected oocytes that were not treated with antisense showed activities attributable to Cx38 hemichannels that were easily distinguishable from Cx35 hemichannels by a significantly larger unitary conductance (gamma(hc): 250-320 pS). The gamma(hc) of Cx35 hemichannels exhibited a pronounced V(m) dependence; i.e., gamma(hc) increased/decreased with relative hyperpolarization/depolarization (gamma(hc) was 72 pS at V(m) = -100 mV and 35 pS at V(m) = 100 mV). Extrapolation to V(m) = 0 mV predicted a gamma(hc) of 48 pS, suggesting a unitary conductance of intact Cx35 gap junction channels of approximately 24 pS. Channel gating was also V(m) dependent: open time declined with negative V(m) and increased with positive V(m). The ability to break down the complex gating of intact intercellular channels into component hemichannels in vitro will help to evaluate putative physiological roles for hemichannels in vivo.

Zebrafish Cx35: cloning and characterization of a gap junction gene highly expressed in the retina.

The vertebrate connexin gene family encodes protein subunits of gap junction channels, which provide a route for direct intercellular communication. Consequently, gap junctions play a vital role in many developmental and homeostatic processes. Aberrant functioning of gap junctions is implicated in many human diseases. Zebrafish are an ideal vertebrate model to study development of the visual system as they produce transparent embryos that develop rapidly, thereby facilitating morphological and behavioral testing. In this study, zebrafish connexin35 has been cloned from a P1 artificial chromosome (PAC) library. Sequence analysis shows a high degree of similarity to the Cx35/36 orthologous group, which are expressed primarily in nervous tissue, including the retina. The gene encodes a 304-amino acid protein with a predicted molecular weight of approximately 35 kDa. Injection of zebrafish Cx35 RNA into paired Xenopus oocytes elicited intercellular electrical coupling with weak voltage sensitivity. In development, Cx35 is first detectable by Northern analysis and RT-PCR, at 2 days post-fertilization (2 dpf), and in the adult it is expressed in the brain and retina. Immunohistochemical analysis revealed that the Cx35 protein is expressed in two sublaminae of the inner plexiform layer of the adult retina. A similar pattern was seen in the 4 and 5 dpf retina, but no labeling was detected in the retina of earlier embryos.