Site-Directed Mutagenesis of the 1,3-ß-Glucan Synthase Catalytic Subunit of Pneumocystis jirovecii and Susceptibility Assays Suggest Its Sensitivity to Caspofungin.

The echinocandin caspofungin inhibits the catalytic subunit Gsc1 of the enzymatic complex synthesizing 1,3-ß-glucan, an essential compound of the fungal wall. Studies with rodents showed that caspofungin is effective against Pneumocystis asci. However, its efficacy against asci of Pneumocystis jirovecii, the species infecting exclusively humans, remains controversial. The aim of this study was to assess the sensitivity to caspofungin of the P. jirovecii Gsc1 subunit, as well as of those of Pneumocystis carinii and Pneumocystis murina infecting, respectively, rats and mice. In the absence of an established in vitro culture method for Pneumocystis species, we used functional complementation of the Saccharomyces cerevisiae gsc1 deletant. In the fungal pathogen Candida albicans, mutations leading to amino acid substitutions in Gsc1 confer resistance to caspofungin. We introduced the corresponding mutations into the Pneumocystis gsc1 genes using site-directed mutagenesis. In spot dilution tests, the sensitivity to caspofungin of the complemented strains decreased with the number of mutations introduced, suggesting that the wild-type enzymes are sensitive. The MICs of caspofungin determined by Etest and YeastOne for strains complemented with Pneumocystis enzymes (respectively, 0.125 and 0.12 µg/ml) were identical to those upon complementation with the enzyme of C. albicans, for which caspofungin presents low MICs. However, they were lower than the MICs upon complementation with the enzyme of the resistant species Candida parapsilosis (0.19 and 0.25 µg/ml). Sensitivity levels of Gsc1 enzymes of the three Pneumocystis species were similar. Our results suggest that P. jirovecii is sensitive to caspofungin during infections, as are P. carinii and P. murina.

Lausannevirus Encodes a Functional Dihydrofolate Reductase Susceptible to Proguanil.

Lausannevirus belongs to the family Marseilleviridae within the group of nucleocytoplasmic large DNA viruses (NCLDVs). These giant viruses exhibit unique features, including a large genome, ranging from 100 kb to 2.5 Mb and including from 150 to more than 2,500 genes, as well as the presence of genes coding for proteins involved in transcription and translation. The large majority of Lausannevirus open reading frames have unknown functions. Interestingly, a bifunctional dihydrofolate reductase-thymidylate synthase (DHFR-TS) is encoded in the Lausannevirus genome. The enzyme plays central roles in DNA precursor biosynthesis. DHFR is the pharmacological target of antifolates, such as trimethoprim, pyrimethamine, and proguanil. First, the functionality of Lausannevirus DHFR-TS was demonstrated by the successful complementation of a DHFR-deficient Saccharomyces cerevisiae strain with a plasmid expressing the heterologous gene. Additionally, using this heterologous expression system, we demonstrated the in vitro susceptibility of Lausannevirus DHFR-TS to proguanil and its resistance to pyrimethamine and trimethoprim. Proguanil may provide a unique and useful treatment if Lausannevirus proves to be a human pathogen. To our knowledge, this is the first time that a DHFR-TS has been described and characterized in an NCLDV.

Prospective multicenter international surveillance of azole resistance in Aspergillus fumigatus.

To investigate azole resistance in clinical Aspergillus isolates, we conducted prospective multicenter international surveillance. A total of 3,788 Aspergillus isolates were screened in 22 centers from 19 countries. Azole-resistant A. fumigatus was more frequently found (3.2% prevalence) than previously acknowledged, causing resistant invasive and noninvasive aspergillosis and severely compromising clinical use of azoles.

Functional characterization of the Pneumocystis jirovecii potential drug targets dhfs and abz2 involved in folate biosynthesis.

Pneumocystis species are fungal parasites colonizing mammal lungs with strict host specificity. Pneumocystis jirovecii is the human-specific species and can turn into an opportunistic pathogen causing severe pneumonia in immunocompromised individuals. This disease is currently the second most frequent life-threatening invasive fungal infection worldwide. The most efficient drug, cotrimoxazole, presents serious side effects, and resistance to this drug is emerging. The search for new targets for the development of new drugs is thus of utmost importance. The recent release of the P. jirovecii genome sequence opens a new era for this task. It can now be carried out on the actual targets to be inhibited instead of on those of the relatively distant model Pneumocystis carinii, the species infecting rats. We focused on the folic acid biosynthesis pathway because (i) it is widely used for efficient therapeutic intervention, and (ii) it involves several enzymes that are essential for the pathogen and have no human counterparts. In this study, we report the identification of two such potential targets within the genome of P. jirovecii, the dihydrofolate synthase (dhfs) and the aminodeoxychorismate lyase (abz2). The function of these enzymes was demonstrated by the rescue of the null allele of the orthologous gene of Saccharomyces cerevisiae.

A Pneumocystis jirovecii pneumonia outbreak in a single kidney-transplant center: role of cytomegalovirus co-infection.

Pneumocystis jirovecii pneumonia (PCP) and cytomegalovirus (CMV) infection represent possible complications of medical immunosuppression. Between 2005 and 2010, non-human immunodeficiency virus (HIV) PCP patients admitted to a nephrology unit were analyzed for outcome, CMV comorbidity, and patient-to-patient contacts prior to PCP. In contrast to 2002-2004 (no cases) and 2008-2010 (10 cases), a PCP outbreak of 29 kidney-transplant recipients and one patient with anti-glomerular basement membrane disease occurred between 2005 and 2007. None of the patients were on PCP chemoprophylaxis. In four PCP patients, the genotyping data of bronchoalveolar lavage specimen showed an identical Pneumocystis strain. PCP cases had a higher incidence of CMV infection (12 of 30 PCP patients) and CMV disease (four patients) when compared to matched PCP-free controls (p < 0.05). Cotrimoxazole and, if applicable, ganciclovir were started 2.0 ± 4.0 days following admission, and immunosuppressive medication was reduced. In-hospital mortality was 10% and the three-year mortality was 20%. CMV co-infection did not affect mortality. CMV co-infection more frequently occurred during a cluster outbreak of non-HIV PCP in comparison to PCP-free controls. Here, CMV awareness and specific therapy of both CMV infection and PCP led to a comparatively favorable patient outcome. The role of patient isolation should be further investigated in incident non-HIV PCP.

[Coronary CT scanning in routine clinical practice]. / Le CT-scan des artères coronaires en pratique clinique courante.

Cardiac CT scanning is the only non-invasive test capable of defining coronary anatomy and evaluating the degree and severity of coronary stenoses. We studied its application at La Tour hospital in Geneva: 108 patients had a cardiac CT scan in 2009. The main indication was the investigation of chest pain in patients with an intermediate risk profile. We confirmed that cardiac CT scanning tends to overestimate lesion severity, particularly when heavy calcification is present but has an excellent negative predictive value. Thanks to decreasing radiation exposure with new protocols and machines, cardiac CT scanning is fast becoming a useful option to evaluate coronary disease, especially when the pre-test probability is intermediate and/or functional testing is non conclusive.

Site-specific alterations in the B oligomer that affect receptor-binding activities and mitogenicity of pertussis toxin.

Pertussis toxin plays a major role in the pathogenesis of whooping cough and is considered an important constituent of vaccines against this disease. It is composed of five different subunits associated in a molar ratio 1S1:1S2:1S3:2S4:1S5. The S1 subunit is responsible for the ADP-ribosyltransferase activity of the toxin. The B moiety, composed of S2 through S5, recognizes and binds to the target cell receptors and has some ADP-ribosyltransferase-independent activities such as mitogenicity. Site-directed mutagenesis of subunits S2 and S3 allowed us to identify amino acid residues involved in receptor binding. Of all the modifications generated, the deletion of Asn 105 in S2 and of Lys 105 in S3 resulted in the more drastic reduction of binding to haptoglobin and CHO cells, respectively. A holotoxin carrying both deletions presented a mitogenicity reduced to an undetectable level. The combination of these B oligomer mutations with two substitutions in the S1 subunit led to the production of a toxin analog with reduced ADP-ribosyltransferase-dependent and -independent activities including mitogenicity. As shown by immunoprecipitation with various monoclonal antibodies, the mutant holotoxin was correctly assembled and antigenically similar to the native toxin. This toxin analog induced toxin-neutralizing antibodies at the same level as the holotoxin carrying only mutations in the S1 subunit, and may therefore be considered a useful candidate for the development of a new generation vaccine against whooping cough.

Molecular evidence of interhuman transmission in an outbreak of Pneumocystis jirovecii pneumonia among renal transplant recipients.

Pneumocystis jirovecii pneumonia (PCP) remains an important cause of morbidity and mortality in immunocompromised individuals. The epidemiology and pathogenesis of this infection are poorly understood, and the exact mode of transmission remains unclear. Recent studies reported clusters of PCP among immunocompromised patients, raising the suspicion of interhuman transmission. An unexpected increase of the incidence of PCP cases in our nephrology outpatient clinic prompted us to conduct a detailed analysis. Genotyping of 7 available specimens obtained from renal transplant recipients was performed using multi-locus DNA sequence typing (MLST). Fragments of 4 variable regions of the P. jirovecii genome (ITS1, 26S, mt26S, beta-tubulin) were sequenced and compared with those of 4 independent control patients. MLST analysis revealed identical sequences of the 4 regions among all 7 renal allograft recipients with available samples, indicating an infection with the same P. jirovecii genotype. We observed that all but 1 of the 19 PCP-infected transplant recipients had at least 1 concomitant visit with another PCP-infected patient within a common waiting area. This study provides evidence that nosocomial transmission among immunocompromised patients may have occurred in our nephrology outpatient clinic. Our findings have epidemiological implications and suggest that prolonged chemoprophylaxis for PCP may be warranted in an era of more intense immunosuppression.

Duplication of 7q34 is specific to juvenile pilocytic astrocytomas and a hallmark of cerebellar and optic pathway tumours.

BACKGROUND: Juvenile pilocytic astrocytomas (JPA), a subgroup of low-grade astrocytomas (LGA), are common, heterogeneous and poorly understood subset of brain tumours in children. Chromosomal 7q34 duplication leading to fusion genes formed between KIAA1549 and BRAF and subsequent constitutive activation of BRAF was recently identified in a proportion of LGA, and may be involved in their pathogenesis. Our aim was to investigate additional chromosomal unbalances in LGA and whether incidence of 7q34 duplication is associated with tumour type or location. METHODS AND RESULTS: Using Illumina-Human-Hap300-Duo and 610-Quad high-resolution-SNP-based arrays and quantitative PCR on genes of interest, we investigated 84 paediatric LGA. We demonstrate that 7q34 duplication is specific to sporadic JPA (35 of 53 - 66%) and does not occur in other LGA subtypes (0 of 27) or NF1-associated-JPA (0 of 4). We also establish that it is site specific as it occurs in the majority of cerebellar JPA (24 of 30 - 80%) followed by brainstem, hypothalamic/optic pathway JPA (10 of 16 - 62.5%) and is rare in hemispheric JPA (1 of 7 - 14%). The MAP-kinase pathway, assessed through ERK phosphorylation, was active in all tumours regardless of 7q34 duplication. Gain of function studies performed on hTERT-immortalised astrocytes show that overexpression of wild-type BRAF does not increase cell proliferation or baseline MAPK signalling even if it sensitises cells to EGFR stimulation. CONCLUSIONS AND INTERPRETATION: Our results suggest that variants of JPA might arise from a unique site-restricted progenitor cell where 7q34 duplication, a hallmark of this tumour-type in association to MAPK-kinase pathway activation, potentially plays a site-specific role in their pathogenesis. Importantly, gain of function abnormalities in components of MAP-Kinase signalling are potentially present in all JPA making this tumour amenable to therapeutic targeting of this pathway.

Analytical study of microsomes and isolated subcellular membranes from rat liver. V. Immunological localization of cytochrome b5 by electron microscopy: methodology and application to various subcellular fractions.

The localization of cytochrome b5 on the membranes of various subcellular organelles of rat liver was studied by a cytoimmunological procedure using anti-cytochrome b5/anti-ferritin hybrid antibodies and ferritin as label. For this study, highly purified and biochemically characterized membrane preparations were employed. Outer mitochondrial membranes were found to be heavily labeled by the hybrid antibodies whereas Golgi and plasma membranes were not marked by the reagent. Peroxisome membranes were moderately labeled by the hybrid antibodies, suggesting that they may contain some cytochrome b5. The preparation and purification of hybrid antibodies without peptic digestion is described and an analysis made of the composition of the final reagent product.

Evaluation of a polymerase chain reaction-restriction fragment length polymorphism assay for dermatophyte and nondermatophyte identification in onychomycosis.

BACKGROUND: Dermatophytes are the main cause of onychomycoses, but various nondermatophyte filamentous fungi are often isolated from abnormal nails. The correct identification of the aetiological agent of nail infections is necessary in order to recommend appropriate treatment. OBJECTIVE: To evaluate a rapid polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay based on 28S rDNA for fungal identification in nails on a large number of samples in comparison with cultures. METHODS: Infectious fungi were analysed using PCR-RFLP in 410 nail samples in which fungal elements were observed in situ by direct mycological examination (positive samples). The results were compared with those previously obtained by culture of fungi on Sabouraud agar from the same nail samples. RESULTS: PCR-RFLP identification of fungi in nails allowed validation of the results obtained in culture when Trichophyton spp. grew from infected samples. In addition, nondermatophyte filamentous fungi could be identified with certainty as the infectious agents in onychomycosis, and discriminated from dermatophytes as well as from transient contaminants. The specificity of the culture results relative to PCR-RFLP appeared to be 81%, 71%, 52% and 63% when Fusarium spp., Scopulariopsis brevicaulis, Aspergillus spp. and Candida spp., respectively, grew on Sabouraud agar. It was also possible to identify the infectious agent when direct nail mycological examination showed fungal elements, but negative results were obtained from fungal culture. CONCLUSIONS: Improved sensitivity for the detection of fungi in nails was obtained using the PCR-RFLP assay. Rapid and reliable molecular identification of the infectious fungus can be used routinely and presents several important advantages compared with culture in expediting the choice of appropriate antifungal therapy.

Encoding, rehearsal, and recall in signers and speakers: shared network but differential engagement.

Short-term memory (STM), or the ability to hold verbal information in mind for a few seconds, is known to rely on the integrity of a frontoparietal network of areas. Here, we used functional magnetic resonance imaging to ask whether a similar network is engaged when verbal information is conveyed through a visuospatial language, American Sign Language, rather than speech. Deaf native signers and hearing native English speakers performed a verbal recall task, where they had to first encode a list of letters in memory, maintain it for a few seconds, and finally recall it in the order presented. The frontoparietal network described to mediate STM in speakers was also observed in signers, with its recruitment appearing independent of the modality of the language. This finding supports the view that signed and spoken STM rely on similar mechanisms. However, deaf signers and hearing speakers differentially engaged key structures of the frontoparietal network as the stages of STM unfold. In particular, deaf signers relied to a greater extent than hearing speakers on passive memory storage areas during encoding and maintenance, but on executive process areas during recall. This work opens new avenues for understanding similarities and differences in STM performance in signers and speakers.

Expression and Immunostaining Analyses Suggest that Pneumocystis Primary Homothallism Involves Trophic Cells Displaying Both Plus and Minus Pheromone Receptors.

The genus Pneumocystis encompasses fungal species that colonize mammals' lungs with host specificity. Should the host immune system weaken, the fungal species can cause severe pneumonia. The life cycle of these pathogens is poorly known, mainly because an in vitro culture method has not been established. Both asexual and sexual cycles would occur. Trophic cells, the predominant forms during infection, could multiply asexually but also enter into a sexual cycle. Comparative genomics revealed a single mating type locus, including plus and minus genes, suggesting that primary homothallism involving self-fertility of each strain is the mode of reproduction of Pneumocystis species. We identified and analyzed the expression of the mam2 and map3 genes encoding the receptors for plus and minus pheromones using reverse transcriptase PCR, in both infected mice and bronchoalveolar lavage fluid samples from patients with Pneumocystis pneumonia. Both receptors were most often concomitantly expressed during infection, revealing that both pheromone-receptor systems are involved in the sexual cycle. The map3 transcripts were subject to alternative splicing. Using immunostaining, we investigated the presence of the pheromone receptors at the surfaces of Pneumocystis cells from a patient. The staining tools were first assessed in Saccharomyces cerevisiae displaying the Pneumocystis receptors at their cellular surface. Both receptors were present at the surfaces of the vast majority of the cells that were likely trophic forms. The receptors might have a role in mate recognition and/or postfertilization events. Their presence at the cell surface might facilitate outbreeding versus inbreeding of self-fertile strains.IMPORTANCE The fungi belonging to the genus Pneumocystis may cause severe pneumonia in immunocompromised humans, a disease that can be fatal if not treated. This disease is nowadays one of the most frequent invasive fungal infections worldwide. Whole-genome sequencing revealed that the sexuality of these fungi involves a single partner that can self-fertilize. Here, we report that two receptors recognizing specifically excreted pheromones are involved in this self-fertility within infected human lungs. Using fluorescent antibodies binding specifically to these receptors, we observed that most often, the fungal cells display both receptors at their surface. These pheromone-receptor systems might play a role in mate recognition and/or postfertilization events. They constitute an integral part of the Pneumocystis obligate sexuality within human lungs, a cycle that is necessary for the dissemination of the fungus to new individuals.

Nosocomial Pneumocystis jirovecii infections.

Airborne transmission of Pneumocystis sp. from host to host has been demonstrated in rodent models and several observations suggest that interindividual transmission occurs in humans. Moreover, it is accepted that the Pneumocystis organisms infecting each mammalian species are host specific and that the hypothesis of an animal reservoir for Pneumocystis jirovecii (P. jirovecii), the human-specific Pneumocystis species, can be excluded. An exosaprophytic form of the fungus cannot be strictly ruled out. However, these data point toward the potential for the specific host to serve as its own reservoir and for Pneumocystis infection in humans as an anthroponosis with humans as a reservoir for P. jirovecii. This review highlights the main data on host-to-host transmission of Pneumocystis in rodent models and in humans by the airborne route and provides a rationale for considering the occurrence of nosocomial infections and measures for their prevention

Functional and Expression Analyses of the Pneumocystis MAT Genes Suggest Obligate Sexuality through Primary Homothallism within Host Lungs.

Fungi of the genus Pneumocystis are obligate parasites that colonize mammals' lungs and are host species specific. Pneumocystis jirovecii and Pneumocystis carinii infect, respectively, humans and rats. They can turn into opportunistic pathogens in immunosuppressed hosts, causing severe pneumonia. Their cell cycle is poorly known, mainly because of the absence of an established method of culture in vitro It is thought to include both asexual and sexual phases. Comparative genomic analysis suggested that their mode of sexual reproduction is primary homothallism involving a single mating type (MAT) locus encompassing plus and minus genes (matMc, matMi, and matPi; Almeida et al., mBio 6:e02250-14, 2015). Thus, each strain would be capable of sexual reproduction alone (self-fertility). However, this is a working hypothesis derived from computational analyses that is, in addition, based on the genome sequences of single isolates. Here, we tested this hypothesis in the wet laboratory. The function of the P. jirovecii and P. carinii matMc genes was ascertained by restoration of sporulation in the corresponding mutant of fission yeast. Using PCR, we found the same single MAT locus in all P. jirovecii isolates and showed that all three MAT genes are often concomitantly expressed during pneumonia. Extensive homology searches did not identify other types of MAT transcription factors in the genomes or cis-acting motifs flanking the MAT locus that could have been involved in MAT switching or silencing. Our observations suggest that Pneumocystis sexuality through primary homothallism is obligate within host lungs to complete the cell cycle, i.e., produce asci necessary for airborne transmission to new hosts.IMPORTANCE Fungi of the genus Pneumocystis colonize the lungs of mammals. In immunosuppressed human hosts, Pneumocystis jirovecii may cause severe pneumonia that can be fatal. This disease is one of the most frequent life-threatening invasive fungal infections in humans. The analysis of the genome sequences of these uncultivable pathogens suggested that their sexual reproduction involves a single partner (self-fertilization). Here, we report laboratory experiments that support this hypothesis. The function of the three genes responsible for sexual differentiation was ascertained by the restoration of sexual reproduction in the corresponding mutant of another fungus. As predicted by self-fertilization, all P. jirovecii isolates harbored the same three genes that were often concomitantly expressed within human lungs during infection. Our observations suggest that the sexuality of these pathogens relies on the self-fertility of each isolate and is obligate within host lungs to complete the cell cycle and allow dissemination of the fungus to new hosts.

Differential expression of mutant and normal beta T3 receptor alleles in kindreds with generalized resistance to thyroid hormone.

Thyroid hormone resistance (THR) is primarily an autosomal dominant inherited disease characterized by resistance of pituitary and peripheral tissues to the action of thyroid hormone. We investigated whether the heterogeneous phenotypic features that occur not only among kindreds but also within the same kindred might be due to the expression of differing ratios of mutant and normal receptors in tissues. Using an allele-specific primer extension method, we determined the relative expression of normal and mutant mRNAs from the fibroblasts of affected and unaffected members of two kindreds with TRH: A-H and N-N. While two affected members of A-H, as expected, had nearly equal amounts of normal and mutant hTR beta mRNA, two other members had mutant mRNA levels that accounted for at least 70% of the hTR beta mRNA. Phenotypic variability within and between kindreds with generalized resistance to thyroid hormone GRTH may be due to this differential expression of the mutant and wild type mRNA. Furthermore, when several clinical parameters of THR were compared in several affected members from two kindreds with GRTH, we found that two cases in one kindred exhibited a high mutant-to-normal hTR beta ratio and had considerably more bone resistance during their development. In certain kindreds with THR, differing ratios of normal and mutant hTR receptors may be age and growth related and may account for the reported attenuation of phenotypic symptoms with age.

Role of a pituitary-specific transcription factor (pit-1/GHF-1) or a closely related protein in cAMP regulation of human thyrotropin-beta subunit gene expression.

cAMP regulation of the human thyrotropin-beta (TSH beta) gene cAMP was studied in two heterologous cell lines, a human embryonal kidney cell line (293) and a rat pituitary cell line (GH3). In 293 cells, human TSH beta gene expression was not stimulated by the adenylate cyclase activator forskolin or the cAMP analogue 8-bromo-cAMP (8-Br-cAMP). On the other hand, these agents induced human TSH beta gene expression 4-12-fold in GH3 cells. Deletion analysis demonstrated that the regions from +3 to +8 bp and from -128 to -61 bp were both necessary for cAMP stimulation. The latter region contains three DNA sequences homologous to a pituitary-specific transcription factor, Pit-1/GHF-1, DNA-binding site. Gel-mobility assays demonstrated that a radiolabeled human TSH beta probe (-128 to -61 bp) formed five specific DNA-protein complexes with mouse thyrotropic tumor (MTT) nuclear extract and two specific complexes with in vitro translated Pit-1/GHF-1. Four of the five MTT complexes and both in vitro Pit-1/GHF-1 complexes were reduced or eliminated by excess of an unlabeled Pit-1/GHF-1 DNA-binding site from the rat growth hormone gene, but not a mutated version of the same DNA fragment, suggesting that Pit-1/GHF-1 or a closely related thyrotroph protein binds to these DNA sequences. In 293 cells, co-transfection of an expression vector containing the Pit-1/GHF-1 cDNA restored cAMP-responsiveness to the human TSH beta promoter (5.2- and 6.6-fold maximal stimulation by 8-Br-cAMP and forskolin, respectively) but not the herpes virus thymidine kinase promoter (1.2-fold maximal stimulation by either agent). Thus we conclude that the human TSH beta gene is positively regulated by cAMP in GH3 but not 293 cells. Since the human TSH beta gene contains at least one high-affinity binding site for Pit-1/GHF-1 in a region necessary for cAMP stimulation and cAMP stimulation could be restored to the human TSH beta promoter in a previously nonresponsive cell line by the addition of Pit-1/GHF-1, this suggests that Pit-1/GHF-1, or a closely related protein in the thyrotroph, may be a trans-acting factor for cAMP stimulation of the TSH beta gene.

Loss of the common "A" determinant of hepatitis B surface antigen by a vaccine-induced escape mutant.

A previous study (Carman, W. F., A. R. Zanetti, P. Karayiannis, J. A. Waters, G. Manzillo, E. Tanzi, A. J. Zuckerman, and H. C. Thomas. 1990. Lancet. 336:325-329) demonstrated a variant hepatitis B surface antigen (HBsAg) in a vaccinated child born to a hepatitis B virus-infected mother. A substitution of arginine for glycine at amino acid 145 in HBsAg was observed. In this study the effect of this substitution on the common "a" determinant of this protein, against which protective immunity is directed, is investigated. Using recombinant HBsAg with and without the amino acid substitution, the binding of monoclonal antibodies that recognize different epitopes of the "a" determinant, was shown to be destroyed by the presence of arginine at amino acid 145. In convalescent and vaccinee sera, antibody binding to HBsAg was not inhibited by the variant HBsAg. Immunization with the variant HBsAg, although eliciting a high titer antibody that recognized the variant, produced a low titer of antibody recognizing the native protein. Studies in mice demonstrate that the immunogenicity of the variant protein is also substantially altered. The data presented here demonstrate that this variant evades the known protective anti-HBs response and lends support to the suggestion that this mutation arose as the result of immune pressure.

A base mutation of the C-erbA beta thyroid hormone receptor in a kindred with generalized thyroid hormone resistance. Molecular heterogeneity in two other kindreds.

Generalized thyroid hormone resistance (GTHR) is a disorder of thyroid hormone action that we have previously shown to be tightly linked to one of the two thyroid hormone receptor genes, c-erbA beta, in a single kindred, A. We now show that in two other kindreds, B and D, with differing phenotypes, there is also linkage between c-erbA beta and GTHR. The combined maximum logarithm of the odds score for all three kindreds at a recombination fraction of 0 was 5.77. In vivo studies had shown a triiodothyronine (T3)-binding affinity abnormality in nuclear receptors of kindred A, and we therefore investigated the defect in c-erbA beta in this kindred by sequencing a major portion of the T3-binding domain in the 3'-region of fibroblast c-erbA beta cDNA and leukocyte c-erbA beta genomic DNA. A base substitution, cytosine to adenine, was found at cDNA position 1643 which altered the proline codon at position 448 to a histidine. By allelic-specific hybridization, this base substitution was found in only one allele of seven affected members, and not found in 10 unaffected members of kindred A, as expected for a dominant disease. Also, this altered base was not found in kindreds B or D, or in 92 random c-erbA beta alleles. These results and the fact that the mutation is predicted to alter the secondary structure of the crucial T3-binding domain of the c-erbA beta receptor suggest this mutation is an excellent candidate for the genetic cause of GTHR in kindred A. Different mutations in the c-erbA beta gene are likely responsible for the variant phenotypes of thyroid hormone resistance in kindreds B and D.