Follistatin is a candidate endogenous negative regulator of activin A in experimental allergic asthma.

BACKGROUND: Activin A is a member of the transforming growth factor-beta superfamily which is directly implicated in airway structural change and inflammation in asthma. In vitro, the biological effects of activin A are neutralized by the soluble binding protein follistatin. OBJECTIVE: To determine the potential of endogenous follistatin to suppress activin A in vivo by analysing their relative tissue and kinetic compartmentalization during the effector phase of subchronic Th2-driven mucosal inflammation in a murine model of allergic asthma. METHODS: Eosinophilic mucosal inflammation was elicited by triggering Th2 recall responses by antigen challenge in ovalbumin-sensitized BALB/c mice. The kinetics and distribution of activin A and follistatin protein were assessed in lung tissue and bronchoalveolar lavage fluid and measured in relation to airway eosinophilia, goblet cell metaplasia and Th2 cytokine production in mediastinal lymph nodes. RESULTS: Follistatin was released concurrently with activin A suggesting it acts as an endogenous regulator: peak BAL concentrations coincided with maximal airway eosinophilia, and frequency of IL-4, IL-5 and IL-13 producing cells in mediastinal lymph nodes but induction lagged behind the onset of inflammation. Follistatin and activin A immunoreactivity were lost in airway epithelial cells in parallel with goblet cell metaplasia. Exogenous follistatin inhibited the allergen-specific Th2 immune response in mediastinal lymph nodes and mucus production in the lung. CONCLUSION: Follistatin is preformed in the normal lung and released in concert with activin A suggesting it serves as an endogenous regulator. Disturbance of the fine balance between activin A and its endogenous inhibitor follistatin may be a determinant of the severity of allergic inflammation or tissue phenotypic shift in asthma.

Expression of monocyte chemoattractant protein-1 and macrophage colony-stimulating factor in normal and inflamed rat testis.

Macrophages are numerous in the testicular interstitial tissue under normal conditions and increase during inflammation. The mechanisms involved are poorly characterized. Expression of the macrophage-regulating cytokines monocyte chemoattractant protein (MCP)-1 and macrophage colony-stimulating factor (M-CSF) was examined in the adult rat testis before and after an i.p. injection of an inflammatory stimulus, lipopolysaccharide (LPS). In the normal testis, M-CSF was readily observed using Northern blot and Western blot analysis. In contrast, MCP-1 was not detectable by Northern blot in the normal testis, but was detected using RT-PCR amplification and a sensitive ELISA. After LPS treatment, testicular MCP-1 mRNA and protein expression increased dramatically (up to 400-fold). In-situ hybridization for MCP-1 revealed that production was confined to the interstitium of the inflamed testis, in Leydig cells, peritubular cells, perivascular cells and monocyte-like macrophages, but not in tissue-resident macrophages. Unlike MCP-1, M-CSF mRNA and protein expression in the testis increased only marginally, if at all, after LPS treatment. These results suggest that MCP-1 stimulates the increase in intratesticular macrophages that accompanies LPS-induced inflammation in vivo. Together with M-CSF, MCP-1 may also play a role in maintaining the resident macrophage population of the normal testis.

Tpx-1 is a component of the outer dense fibers and acrosome of rat spermatozoa.

Previously we reported the cloning of a member of the cysteine-rich secretory protein family, tpx-1, from a testis expression library using an outer dense fiber (ODF)-specific antiserum. Using immunohistochemical and immunoelectron microscopic techniques and Western blotting of purified sperm tail components, we have determined that tpx-1 exists as 25 and 27 kDa proteins in two components of rat spermatid: the ODFs and the acrosome. Tpx-1 mRNA is first expressed in the late pachytene spermatocytes, but the production of these tpx-1 proteins is translationally delayed for 4-5 days before being incorporated into the developing sperm acrosome, surrounding the elongating and condensing spermatid nucleus. Concurrent with sperm head formation, tpx-1 protein was incorporated into the developing sperm tail, and specifically the ODFs. The tpx-1 protein was seen within structures resembling granulated bodies in the cytoplasmic lobe of elongating spermatids and was incorporated subsequently into the growing tail in a manner consistent with ODF development. In addition, tpx-1 protein was localized at the ultrastructural level of the connecting piece of the neck and longitudinal columns of the fibrous sheath, suggesting common protein components in these cytoskeletal structures. As such, tpx-1 may have functional significance in the processes of sperm head development and tail function.

Cloning and regulation of the rat activin betaE subunit.

Using a combination of polymerase chain reaction (PCR) procedures, we have cloned and sequenced the rat activin beta(E) subunit cDNA. The putative protein corresponding to the prepro-activin beta(E) subunit was predicted to comprise 350 amino acids which, when cleaved between amino acid residues 236 and 237, would yield a mature polypeptide of approximately M(r) 12 500 with a predicted pI of 5.1. Two cDNA transcripts for activin beta(E) were identified; these differed by 738 bp in the 3'-untranslated region. Activin beta(E) mRNA transcripts were expressed only in rat liver and lung tissue as assessed by Northern blotting and PCR analysis. Relatively higher levels of both transcripts were found in the liver, whereas the lung contained lower levels that were detectable by PCR only. In situ hybridisation data showed that, within the liver, activin beta(E) mRNA was localised to hepatocytes. In vivo treatment with lipopolysaccharide as a means of activating the immune system and the hepatic acute-phase response resulted in stimulated activin beta(E) mRNA levels, compared with untreated, control rats. This increased expression was accompanied by a preferential increase in the amount of the long activin beta(E) transcript over the shorter transcript. These findings suggested that the two activin beta(E) mRNA transcripts may be products of alternative splicing events or use alternative polyadenylation sites which are differentially regulated during inflammation. These data provide evidence of a role for activin beta(E) in liver function and inflammation in the rat.

Stability of human immunodeficiency virus RNA in blood specimens as measured by a commercial PCR-based assay.

We investigated the effects of conditions often encountered during handling, transit, and storage of blood specimens on the quantity of detectable human immunodeficiency virus (HIV) RNA in plasma. HIV RNA copy numbers were measured with a commercially available assay (the Amplicor HIV-1 Monitor test kit). Variables examined were the time to processing of blood and plasma, the holding temperature of blood and plasma prior to processing, the effect of freezing and thawing of plasma, and the use of different anticoagulants. The relationship between the HIV RNA copy number and the HIV isolation rate by peripheral blood mononuclear cell (PBMC) coculture was also examined. We found that RNA copy numbers were maintained to within 0.5 log10 (approximately threefold) in blood and plasma samples held at room temperature or 4 degrees C for up to 3 days and remained stable despite (limited) freezing and thawing of the plasma. HIV RNA copy numbers were also maintained after long-term storage of plasma at -70 degrees C. The ability to isolate HIV from PBMCs was directly proportional to the HIV RNA copy number.

Syncytium-inducing phenotype and zidovudine susceptibility of HIV-1 isolated from post-mortem tissue.

OBJECTIVE: To establish the syncytium-inducing (SI) phenotype and zidovudine (ZDV) susceptibility of HIV-1 isolates obtained from autopsy specimens. METHODS: Isolation of HIV was attempted from autopsy specimens obtained from 76 AIDS patients. Specimens of lymph node, spleen, spinal cord, brain and cerebrospinal fluid (CSF) were processed and cultured with peripheral blood mononuclear cells (PBMC) from seronegative donors. Biological phenotype was determined in a T-lymphocyte line (MT-2). ZDV susceptibility was evaluated in a PBMC-based assay. Sequencing of amino-acid codons in the reverse transcriptase gene previously shown to be associated with ZDV resistance was carried out on a subgroup of isolates. RESULTS: HIV was recovered from tissue specimens and CSF up to 5 days post-mortem, but recovery rate of infectious virus decreased as the time between autopsy and specimen processing increased. There was a lack of concordance between PBMC isolates and isolates from different tissue sites with respect to SI phenotype. ZDV-resistant virus was isolated from post-mortem specimens of patients who had received long-term ZDV therapy up until or shortly before their death. ZDV-sensitive virus re-emerged in the lymph node of patients who ceased treatment several months prior to death. Phenotypically sensitive virus obtained from lymph node tissue of three patients after a relatively short time off ZDV (4-6 months) retained some of the amino-acid substitutions known to be associated with resistance. CONCLUSION: The data suggests that ZDV resistance and re-emergence of sensitive virus does not originate in peripheral cells, and that these cells and tissues are seeded with virus present elsewhere, possibly in the germinal centres of the lymph node.