Pneumocystis carinii causes a distinctive interstitial pneumonia in immunocompetent laboratory rats that had been attributed to "rat respiratory virus".

A prevalent and distinctive infectious interstitial pneumonia (IIP) of immunocompetent laboratory rats was suspected to be caused by a putative virus, termed rat respiratory virus, but this was never substantiated. To study this disease, 2 isolators were independently populated with rats from colonies with endemic disease, which was perpetuated by the regular addition of naive rats. After Pneumocystis was demonstrated by histopathology and polymerase chain reaction (PCR) in the lungs of rats from both isolators and an earlier bedding transmission study, the relationship between Pneumocystis and IIP was explored further by analyzing specimens from 3 contact transmission experiments, diagnostic submissions, and barrier room breeding colonies, including 1 with and 49 without IIP. Quantitative (q) PCR and immunofluorescence assay only detected Pneumocystis infection and serum antibodies in rats from experiments or colonies in which IIP was diagnosed by histopathology. In immunocompetent hosts, the Pneumocystis concentration in lungs corresponded to the severity and prevalence of IIP; seroconversion occurred when IIP developed and was followed by the concurrent clearance of Pneumocystis from lungs and resolution of disease. Experimentally infected immunodeficient RNU rats, by contrast, did not seroconvert to Pneumocystis or recover from infection. qPCR found Pneumocystis at significantly higher concentrations and much more often in lungs than in bronchial and nasal washes and failed to detect Pneumocystis in oral swabs. The sequences of a mitochondrial ribosomal large-subunit gene region for Pneumocystis from 11 distinct IIP sources were all identical to that of P. carinii. These data provide substantial evidence that P. carinii causes IIP in immunocompetent rats.

E7 proteins from high- and low-risk human papillomaviruses bind to TGF-beta-regulated Smad proteins and inhibit their transcriptional activity.

Human papillomaviruses (HPV) infect keratinocytes of skin and mucosa. Persistent infection can lead to the formation of benign tumors. In cases of high-risk HPV, such as HPV16 or 18, these may further progress to cancer. In order to support viral replication in suprabasal keratinocytes, the HPV E7 protein employs various strategies to keep keratinocytes in cycle and counteracts anti-proliferative signals from outside. HPV16 E7 can directly interfere with transforming growth factor-beta (TGF-beta) signalling by binding to Smad proteins mediating growth arrest. It has been speculated that this property of HPV16 E7 contributes to HPV-associated carcinogenesis. Here, we show that E7 proteins from different low- and high-risk HPV types bind to Smad 1 to 4. The E7 protein from HPV1, a low-risk HPV causing plantar warts, efficiently inhibited Smad 3-induced transcription. Our data strongly indicate that the Smad-binding capacity of E7 proteins from different HPVs may preserve keratinocyte proliferation required for the productive viral life cycle rather than promoting carcinogenesis.

Detection of Leishmania causing visceral leishmaniasis in the Old and New Worlds by a polymerase chain reaction assay based on telomeric sequences.

We present a new polymerase chain reaction assay based on telomeric sequences of Leishmania donovani. When this assay was used in dilutions of purified L. donovani DNA, a strong amplification signal was observed with 1 fg of DNA. In a specificity test that used purified DNA from Old World and New World Leishmania, the assay recognized all parasites isolated from patients with visceral leishmaniasis, except for 2 isolates of Leishmania colombiensis from Venezuela and 1 isolate from Brazil. All Leishmania major and Leishmania tropica isolates tested were negative, except for one isolate in each species. We also used the assay on fresh and archive bone marrow samples recovered from Giemsa-stained slides and from dried blood stains.

Immunization with recombinant LD1 antigens protects against experimental leishmaniasis.

The genes, ORFF and BT1 (previously ORFG), are part of the multigenic LD1 locus on chromosome 35 which is frequently amplified in Leishmania. BT1 encodes a biopterin transporter, while the function of the ORFF gene product is unknown, but it is localized to the nucleus. We show here that immunization of mice with recombinant ORFF and BT1 proteins, individually, or in combination, conferred partial protection against challenge with Leishmania donovani. Protection correlated with the production of antigen-specific antibodies and in vitro splenocyte proliferation. Thus, these antigens can be potential vaccine candidates against visceral leishmaniasis.

A cDNA from the green alga Spermatozopsis similis encodes a protein with homology to the newly discovered Roadblock/LC7 family of dynein-associated proteins.

A clone, designated as B15, was isolated from a cDNA library of the unicellular green alga Spermatozopsis similis and characterised. The deduced amino acid sequence of its open reading frame exhibits high homology to members of the recently discovered roadblock/LC7 protein family (robl/LC7) of dynein-associated proteins. Homologies were highest to a robl/LC7-member from human testis (86%, identity 56%) and to the roadblock protein of Drosophila (88%, identity 52%). Data bank analyses revealed no homologies to known higher plant proteins. B15 is a single copy gene in the genome of Sperm-latozopsis and its transcript was detectable throughout the cell cycle.

The Leishmania donovani LD1 locus gene ORFG encodes a biopterin transporter (BT1).

We have previously described two genes, ORFF and ORFG, from the LD1 locus near one telomere of chromosome 35, which are frequently amplified in Leishmania isolates. In Leishmania donovani LSB-51.1, gene conversion of the rRNA gene locus on chromosome 27 with these two genes resulted in their over-expression, because of their transcription by the RNA polymerase I-mediated rRNA promoter. The predicted ORFG protein has substantial sequence homology to the ESAG10 gene product from the Trypanosoma brucei VSG expression site and both are putative membrane proteins. Using successive rounds of gene replacement of the three ORFG genes in L. donovani LSB-51.1, ORFG null mutants were obtained. These mutant cell lines show a direct relationship between ORFG mRNA, protein expression levels and active transport of biopterin into the cells. Transformation of the null mutant with a plasmid containing ORFG restores biopterin transport activity. In addition, the null mutants are unable to grow in the absence of supplemental biopterin. Thus, ORFG encodes a biopterin transporter and has been renamed BTI.

Serodiagnosis of leishmaniasis with recombinant ORFF antigen.

The serodiagnostic potential of recombinant ORFF protein (rORFF) from Leishmania infantum was assessed by ELISA. Of 49 sera from confirmed cases of visceral leishmaniasis (VL), all were seropositive using 5 ng of rORFF and serum diluted 1:20, while only 38 were positive with 500 ng of soluble antigen (SA) and 44 were positive by a direct agglutination test. There was also a positive correlation between spleen size and level of seropositivity with rORFF or SA. The reciprocal endpoint titer with rORFF was 1,280 for sera from VL patients, but < 20 with sera from malaria, filariasis, and tuberculosis patients, as well as with sera from healthy individuals from endemic and non-endemic areas. Sera from 10 confirmed cutaneous leishmaniasis cases from Turkey were negative or only weakly positive with rORFF although 9 were positive with SA. Thus, rORFF protein appears useful as a sensitive reagent for the differential diagnosis of VL caused by the Leishmania donovani complex.

Early experience and the consumption of alcohol by adult C57BL/6J mice.

Two types of early experience were examined for their effect on voluntary alcohol consumption by adult C57BL/6J mice: the experiences associated with belonging to a particular litter, and the experience of early postweaning choice between water and a 10% alcohol solution. Males from identified litters were individually caged from arrival at three weeks of age and given a choice between 10% alcohol and water when eight weeks old. Another group without notation of litter was given alcohol-water choice upon arrival at three weeks of age. Alcohol intake was examined by three measures: daily licks of 10% alcohol, alcohol selection (percent alcohol drinking), and volume of alcohol drunk daily. Belonging to a particular litter did affect body weight and growth, but had no effect on adult consumption of alcohol. Postweaning exposure to alcohol choice, however, produced a small but significant and prolonged increase in alcohol consumption by adults. Furthermore, a developmental trend was found in mice offered alcohol choice at an early age: alcohol preference developed as postweaning growth slowed.

Implications of methadone maintenance for theories of narcotic addiction.

Clinical success in rehabilitation of heroin addicts with maintenance treatment requires stability of the blood level in a pharmacologically effective range (optimally, 150 to 600 ng/mL)-a phenomenon that emphasizes the central importance of narcotic receptor occupation. It is postulated that the high rate of relapse of addicts after detoxification from heroin use is due to persistent derangement of the endogenous ligand-narcotic receptor system and that methadone in an adequate daily dose compensates for this defect. Some patients with long histories of heroin use and subsequent rehabilitation on a maintenance program do well when the treatment is terminated. The majority, unfortunately, experience a return of symptoms after maintenance is stopped. The treatment, therefore, is corrective but not curative for severely addicted persons. A major challenge for future research is to identify the specific defect in receptor function and to repair it. Meanwhile, methadone maintenance provides a safe and effective way to normalize the function of otherwise intractable narcotic addicts.