Antiangiogenic effect of erythromycin: an in vitro model of Bartonella quintana infection

BACKGROUND: Bartonella quintana, the etiological agent of bacillary angiomatosis (BA), causes endothelial cell proliferation. Erythromycin has dramatic effects on BA, and the effects are largely unexplained by the compound's bacteriostatic properties. Our aim here was to evaluate the possibility that erythromycin alters angiogenesis. METHODS: The effect of erythromycin on B. quintana-induced endothelial cell proliferation was studied using a wild-type strain and an erythromycin-resistant B. quintana mutant. The latter was generated by serial subcultures on blood agar plates. RESULTS: We show that erythromycin significantly inhibits the proliferation of dermal microvascular endothelial cells induced either by wild-type B. quintana or by our erythromycin-resistant mutant. Doxycycline and gentamycin failed to exert such an effect. Finally, we found that the resistant strain harbored a 27-bp insertion in the highly conserved region of the gene encoding the ribosomal protein L4; this insertion may explain the existence of the resistance to erythromycin. CONCLUSION: The data presented here indicate that erythromycin profoundly down-modulates endothelial cell proliferation irrespective of its bacteriostatic effects and suggest that this may be a key component of the efficacy of the compound in the treatment of patients with BA.     

A double-blind low dose-finding phase II study of granulocyte colony-stimulating factor combined with chemotherapy for stem cell mobilization in patients with non-Hodgkin's lymphoma

The aim of the study was to define the minimal effective dose (MED) of granulocyte colony-stimulating factor (G-CSF) among five daily doses following chemotherapy for peripheral blood stem cell (PBSC) collection. Twenty-five patients were included in this double-blind dose-finding phase II study conducted according to a two-stage Bayesian design. The estimated probabilities of success for PBSC collection for the G-CSF doses of 50, 75, 100, 125 and 150 microg/m2/day were 84%, 87.7%, 91%, 93.9 and 96.4%, respectively. Low G-CSF doses may be used with a similar probability of success as conventional doses and could allow significant savings.     

Impaired hair follicle morphogenesis and cycling with abnormal epidermal differentiation in nackt mice,a cathepsin L-deficient mutation

We previously described an autosomal-recessive mutation named nackt (nkt) exhibiting partial alopecia associated with CD4(+) T-cell deficiency. Also, we recently reported that nkt (now Ctsl(nkt)) comprises a deletion in the cathepsin L (Ctsl) gene. Another recent study reported that Ctsl knockout mice have CD4(+) T-cell deficiency and periodic shedding of hair, which recapitulate the nkt mutation and the old furless (fs) mutation. The current study focuses on the dermatological aspects of the nkt mutation. Careful histological analysis of skin development of homozygous nkt mice revealed a delayed hair follicle morphogenesis and late onset of the first catagen stage. The skin of Ctsl(nkt)/Ctsl(nkt) mice showed mild epidermal hyperplasia and hyperkeratosis, severe hyperplasia of the sebaceous glands, and structural alterations of hair follicles. Epidermal differentiation seems to be affected in nkt skin, with overexpression of involucrin and profilaggrin/filaggrin along with focal areas of keratin 6 expression in the interfollicular epidermis. Severe epidermal hyperplasia, acanthosis, orthokeratosis, and hyperkeratosis were only observed in mice maintained in nonpathogen-free environments. The analysis of Rag2-/- Ctsl(nkt)/Ctsl(nkt) double-mutant mice indicates that the skin defect remains under the absence of T and B cells. This animal model provides in vivo evidence that cysteine protease cathepsin L plays a critical role in hair follicle morphogenesis and cycling, as well as epidermal differentiation.     

22q11.2 duplication syndrome: two new familial cases with some overlapping features with DiGeorge/velocardiofacial syndromes

Twenty-one patients, including our two cases, with variable clinical phenotype, ranging from mild learning disability to severe congenital malformations or overlapping features with DiGeorge/velocardiofacial syndromes (DG/VCFS), have been shown to have a chromosome duplication 22q11 of the region that is deleted in patients with DG/VCFS. The reported cases have been identified primarily by interphase FISH and could have escaped identification and been missed by routine cytogenetic analysis. Here we report on two inherited cases, referred to us, to rule out 22q11 microdeletion diagnosis of VCFS. The first patient was a 2-month-old girl, who presented with cleft palate, minor dysmorphic features including short palpebral fissures, widely spaced eyes, long fingers, and hearing loss. Her affected mother had mild mental retardation and learning disabilities. The second patient was a 7(1/2)-year-old boy with velopharyngeal insufficiency and mild developmental delay. He had a left preauricular tag, bifida uvula, bilateral fifth finger clinodactyly, and bilateral cryptorchidism. His facial features appeared mildly dysmorphic with hypertelorism, large nose, and micro/retrognathia. The affected father had mild mental retardation and had similar facial features. FISH analysis of interphase cells showed three TUPLE1-probe signals with two chromosome-specific identification probes in each cell. FISH analysis did not show the duplication on the initial testing of metaphase chromosomes. On review, band q11.2 was brighter on one chromosome 22 in some metaphase spreads. The paucity of reported cases of 22q11.2 microduplication likely reflects a combination of phenotypic diversity and the difficulty of diagnosis by FISH analysis on metaphase spreads. These findings illustrate the importance of scanning interphase nuclei when performing FISH analysis for any of the genomic disorders.     

Lack of intermediate-affinity interleukin-2 receptor in mice leads to dependence on interleukin-2 receptor α, β and γ chain expression for T cell growth

An interleukin (IL)-4 dependant mouse T cell clone 8.2 derived from an IL-2-dependent T cell line was characterized. As measured by flow cytometric analysis and Northern blotting, it expresses IL-2 receptor β (IL-2Rβ) and γ (IL-2Rγ) chains, but has lost expression of IL-2 receptor α chain (IL-2Rα). To investigate the properties of the mouse IL-2Rβγ complex and the role of IL-2Rα gene expression, this clone was further studied. T cell clone 8.2 has lost the capacity to bind ¹²⁵I-labeled human IL-2 under experimental conditions able to detect intermediate-affinity IL-2R in human cells. Mouse IL-2 is unable to block the binding of mAb TMβ1 to 8.2 cells. Under the same experimental conditions, mouse IL-2 blocks the binding of TMβ1 to C30-1 cells expressing the IL-2αβγ complex. Since TMβ1 recognizes an epitope related to the IL-2 binding site of IL-2Rβ, these results can be taken as a demonstration that mouse IL-2Rβγ does not bind mouse IL-2. Furthermore, T cell clone 8.2 does not proliferate in response to recombinant mouse or human IL-2. On the other hand, T cell transfectant lines expressing heterospecific receptors made of the human IL-2Rβ and mouse IL-2Rγ chains bind ¹²⁵I-labeled human IL-2 and proliferate in response to IL-2. This establishes the difference between mouse and human IL-2Rβ chains. Transfection of T cell clone 8.2 with human IL-2Rα genes restores their capacity to proliferate in response to IL-2. In addition, all transfectants grown in IL-2 express the endogeneous mouse IL-2Rα chain. When grown in IL-4, the endogeneous mouse IL-2Rα gene remains silent in all these transfectants. These results show that, contrary to the human, the mouse does not express an intermediate-affinity IL-2R. Expression of the IL-2Rα gene is therefore required for the formation of the functional IL-2R in mice.     

Ligand-induced autoregulation of IL-2 receptor {alpha} chain expression in murine T cell lines

The IL-2 receptor (IL-2R) is composed of three chains a, ßand . In mice, contrary to the human system, we have previouslydemonstrated that the IL-2Rß complex does not bindIL-2. Therefore, mouse IL-2 response is completely dependenton the expression of the IL-2R gene product. T cell clones expressingmouse IL-2Rß and the human IL-2R transgene have beenstudied. When cells are grown in IL-4, mouse IL-2R is not expressed.However, exposure to IL-2 leads to the expression of the endogenousmurine IL-2R subunit. The T cell line expressing mouse IL-2Rand human IL-2Rß can grow in IL-2 but does not expressendogenous murine IL-2 R. Transfection of these cells with thehuman IL-2R gene restores the capacity to induce murine IL-2R.This result demonstrates that IL-2-IL-2R interactions are requiredfor induction of IL-2R. The kinetics of induction and deinductionof murine IL-2R have been studied using clone 18.III. From negativecells, expression of murine IL-2R is a very slow phenomenon.From cells fully expressing IL-2R, deinduction is a two-stepprocess: after a rapid decrease of IL-2R the cells continueto express, for a long period of time, basal levels of murineIL-2R. When cells expressing basal levels of IL-2R are exposedto IL-2, induction of IL-2R is a very rapid phenomenon. Theautoregulatory loop formed by IL-2-IL-2R therefore displaysdifferent levels of functioning.     

Multiplex PCR targeting tpi (triose phosphate isomerase), tcdA (Toxin A), and tcdB (Toxin B) genes for toxigenic culture of Clostridium difficile

A multiplex PCR toxigenic culture approach was designed for simultaneous identification and toxigenic type characterization of Clostridium difficile isolates. Three pairs of primers were designed for the amplification of (i) a species-specific internal fragment of the tpi (triose phosphate isomerase) gene, (ii) an internal fragment of the tcdB (toxin B) gene, and (iii) an internal fragment of the tcdA (toxin A) gene allowing distinction between toxin A-positive, toxin B-positive (A+B+) strains and toxin A-negative, toxin B-positive (A−B+) variant strains. The reliability of the multiplex PCR was established by using a panel of 72 C. difficile strains including A+B+, A−B−, and A−B+ toxigenic types and 11 other Clostridium species type strains. The multiplex PCR assay was then included in a toxigenic culture approach for the detection, identification, and toxigenic type characterization of C. difficile in 1,343 consecutive human and animal stool samples. Overall, 111 (15.4%) of 721 human samples were positive for C. difficile; 67 (60.4%) of these samples contained A+B+ toxigenic isolates, and none of them contained A−B+ variant strains. Fifty (8%) of 622 animal samples contained C. difficile strains, which were toxigenic in 27 (54%) cases, including 1 A−B+ variant isolate. Eighty of the 721 human stool samples (37 positive and 43 negative for C. difficile culture) were comparatively tested by Premier Toxins A&B (Meridian Bioscience) and Triage C. difficile Panel (Biosite) immunoassays, the results of which were found concordant with toxigenic culture for 82.5 and 92.5% of the samples, respectively. The multiplex PCR toxigenic culture scheme described here allows combined diagnosis and toxigenic type characterization for human and animal C. difficile intestinal infections.