Neotropical cloud forests and páramo to contract and dry from declines in cloud immersion and frost.

Clouds persistently engulf many tropical mountains at elevations cool enough for clouds to form, creating isolated areas with frequent fog and mist. Under these isolated conditions, thousands of unique species have evolved in what are known as tropical montane cloud forests (TMCF) and páramo. Páramo comprises a set of alpine ecosystems that occur above TMCF from about 11° N to 9° S along the Americas continental divide. TMCF occur on all continents and island chains with tropical climates and mountains and are increasingly threatened by climate and land-use change. Climate change could impact a primary feature distinguishing these ecosystems, cloud immersion. But where and in what direction cloud immersion of TMCF and páramo will change with climate are fundamental unknowns. Prior studies at a few TMCF sites suggest that cloud immersion will increase in some places while declining in others. Other unknowns include the extent of deforestation in protected and unprotected cloud forest climatic zones, and deforestation extent compared with projected climate change. Here we use a new empirical approach combining relative humidity, frost, and novel application of maximum watershed elevation to project change in TMCF and páramo for Representative greenhouse gas emissions Concentration Pathways (RCPs) 4.5 and 8.5. Results suggest that in <25-45 yr, 70-86% of páramo will dry or be subject to tree invasion, and cloud immersion declines will shrink or dry 57-80% of Neotropical TMCF, including 100% of TMCF across Mexico, Central America, the Caribbean, much of Northern South America, and parts of Southeast Brazil. These estimates rise to 86% of Neotropical TMCF and 98% of páramo in <45-65 yr if greenhouse gas emissions continue rising throughout the 21st century. We also find that TMCF zones are largely forested, but some of the most deforested areas will undergo the least climate change. We project that cloud immersion will increase for only about 1% of all TMCF and in only a few places. Declines in cloud immersion dominate TMCF change across the Neotropics.

Prevalence and causative fungal species of tinea capitis among schoolchildren in Gabon.

Tinea capitis is endemic among schoolchildren in tropical Africa. The objective was to determine the prevalence of symptomatic tinea capitis in schoolchildren in Gabon. A cross-sectional study was conducted with 454 children aged 4-17 years, attending a rural school and an urban school. The diagnosis of tinea capitis was based on clinically manifest infection, direct microscopic examination using 20% potassium hydroxide (KOH) solution and fungal culture. Based on clinical examination, 105 (23.1%) of 454 children had tinea capitis. Seventy-four (16.3%) children were positive by direct examination (KOH) and/or fungal culture. The prevalence of tinea capitis depended on the school studied and ranged from 20.4% in the urban school with a higher socioeconomic status to 26.3% in the rural school with a lower socioeconomic status. Similarly, the spectrum of causative species varied between the different schools. Taken the schools together, Trichophyton soudanense (29.4%) was the most prominent species, followed by Trichophyton tonsurans (27.9%) and Microsporum audouinii (25.0%). Clinically manifest tinea capitis is endemic among schoolchildren in the Lambaréné region in Gabon. The prevalence of tinea capitis and the causative species depended on the type of school that was investigated.

Diagnosis of common dermatophyte infections by a novel multiplex real-time polymerase chain reaction detection/identification scheme.

BACKGROUND: In the absence of a functional dermatophyte-specific polymerase chain reaction (PCR), current diagnosis of dermatophytoses, which constitute the commonest communicable diseases worldwide, relies on microscopy and culture. This combination of techniques is time-consuming and notoriously low in sensitivity. OBJECTIVES: Recent dermatophyte gene sequence records were used to design a real-time PCR assay for detection and identification of dermatophytes in clinical specimens in less than 24 h. PATIENTS AND METHODS: Two assays based on amplification of ribosomal internal transcribed spacer regions and on the use of probes specific to relevant species and species-complexes were designed, optimised and clinically evaluated. One assay was for detecting the Trichophyton mentagrophytes species complex plus T. tonsurans and T. violaceum. The second assayed for the T. rubrum species complex, Microsporum canis and M. audouinii. RESULTS: The analytical sensitivity of both assays was 0.1 pg DNA per reaction, corresponding to 2.5-3.3 genomes per sample. The protocol was clinically evaluated over 6 months by testing 92 skin, nail and hair specimens from 67 patients with suspected dermatophytosis. Real-time PCR detected and correctly identified the causal agent in specimens from which T. rubrum, T. interdigitale, M. audouinii or T. violaceum grew in culture, and also identified a dermatophyte species in an additional seven specimens that were negative in microscopy and culture. CONCLUSIONS: This highly sensitive assay also proved to have high positive and negative predictive values (95.7% and 100%), facilitating the accurate, rapid diagnosis conducive to targeted rather than empirical therapy for dermatophytoses.

Processing of natural and recombinant CXCR3-targeting chemokines and implications for biological activity.

Chemokines comprise a class of peptides with chemotactic activity towards leukocytes. The potency of different chemokines for the same receptor often varies as a result of differences in primary structure. In addition, post-translational modifications have been shown to affect the effectiveness of chemokines. Although in several studies, natural CXCR3-targeting chemokines have been isolated, detailed information about the proteins and their possible modifications is lacking. Using a combination of liquid chromatography and mass spectrometry we studied the protein profile of CXCR3-targeting chemokines expressed by interferon-gamma-stimulated human keratinocytes. The biological implications of one of the identified modifications was studied in more detail using calcium mobilization and chemotaxis assays. We found that the primary structure of human CXCL10 is different from the generally accepted sequence. In addition we identified a C-terminally truncated CXCL10, lacking the last four amino acids. Native CXCL11 was primarily found in its intact mature form but we also found a mass corresponding to an N-terminally truncated human CXCL11, lacking the first two amino acids FP, indicating that this chemokine is a substrate for dipeptidylpeptidase IV. Interestingly, this same truncation was found when we expressed human CXCL11 in Drosophila S2 cells. The biological activity of this truncated form of CXCL11 was greatly reduced, both in calcium mobilization (using CXCR3 expressing CHO cells) as well as its chemotactic activity for CXCR3-expressing T-cells. It is concluded that detailed information on chemokines at the protein level is important to characterize the exact profile of these chemotactic peptides as modifications can severely alter their biological activity.

Cross reactivity of three T cell attracting murine chemokines stimulating the CXC chemokine receptor CXCR3 and their induction in cultured cells and during allograft rejection.

Recent work identified the murine gene homologous to the human T cell attracting chemokine CXC receptor ligand 11 (CXCL11, also termed I-TAC, SCYB11, ss-R1, H174, IP-9). Here, the biological activity and expression patterns of murine CXCL11 relative to CXCL9 (MIG) and CXCL10 (IP-10/crg-2), the other two CXCR3 ligands, were assessed. Calcium mobilization and chemotaxis experiments demonstrated that murine CXCL11 stimulated murine CXCR3 at much lower doses than murine CXCL9 or murine CXCL10. Murine CXCL11 also evoked calcium mobilization in CHO cells transfected with human CXCR3 and was chemotactic for CXCR3-expressing human T lymphocytes as well as for 300--19 pre-B cells transfected with human or murine CXCR3. Moreover, murine CXCL11 blocked the chemotactic effect of human CXCL11 on human CXCR3 transfectants. Depending on cell type (macrophage-like cells RAW264.7, J774A.1, fetal F20 and adult dermal fibroblasts, immature and mature bone marrow-derived dendritic cells) and stimulus (interferons, LPS, IL-1 beta and TNF-alpha), an up to 10,000-fold increase of CXCL9, CXCL10 and CXCL11 mRNA levels, quantified by real-time PCR, was observed. In vivo, the three chemokines are constitutively expressed in various tissues from healthy BALB/c mice and were strongly up-regulated during rejection of allogeneic heart transplants. Chemokine mRNA levels exceeded those of CXCR3 and IFN-gamma which were induced with similar kinetics by several orders of magnitude.

Human IP-9: A keratinocyte-derived high affinity CXC-chemokine ligand for the IP-10/Mig receptor (CXCR3).

Chemokines and their receptors play a crucial part in the recruitment of leukocytes into inflammatory sites. The CXC chemokines IP-10 and Mig are selective attractants for activated (memory) T cells, the predominant cell type in skin infiltrates in many inflammatory dermatoses. The selectivity for activated T cells can be explained by the fact that both chemokines exert their effects through a common receptor, CXCR3, which is nearly exclusively expressed on activated T cells. The aim of this study was to identify biologically active CXCR3 ligands produced by keratinocytes. To that end, Chinese hamster ovary cells expressing a cDNA encoding CXCR3 were challenged with proteins obtained from interferon-gamma stimulated keratinocytes and subsequently monitored for effects on second messenger systems. By this approach we were able to isolate IP-10 and Mig, and in addition identified a novel highly potent ligand for the CXCR3 receptor, designated interferon-gamma-inducible protein-9, which proved to be chemotactic for activated T cells expressing CXCR3. Protein sequence and mass spectrometric analysis followed by molecular cloning of the cDNA encoding interferon-gamma-inducible protein-9, revealed that interferon-gamma-inducible protein-9 is a CXC chemokine with a molecular mass of 8303 Da. From a GenBank database query it became clear that interferon-gamma-inducible protein-9 is in fact the protein encoded by the cDNA sequence also known as beta-R1, H174 or I-TAC. In situ hybridization experiments showed that interferon-gamma-inducible protein-9 mRNA is expressed by basal layer keratinocytes in a variety of skin disorders, including allergic contact dermatitis, lichen planus, and mycosis fungoides suggesting a functional role for this chemokine in skin immune responses.

Plasmodium Vivax resistente a la Cloroquina en la Consulta Externa Pediátrica de Villavicencio

Se revisaron las hostirias clínicas de 671 pacientes quienes asistieron a la consulta externa de pediatría y presentaron como diagnóstico malaria, en un período de 36 meses comprendido entre el 1 de enero de 1993 y 31 de diciembre de 1995. El diagnóstico de malaria se llevó a cabo por parasitemia positiva en gota gruesa y/o extendido de sangre periférica mediante la coloración de Field. Se hallaron 166 pacientes quienes cursaron con malaria por Plasmodium falciparum y 505 por Plasmo-dium vivax de los cuales 9 presentaron resistencia clínica al tratamiento con cloroquina, para lo cual nos basamos en los criterios propuestos por la Organización Mundial de la Salud en 1967. Se analizó en estos 9 pacientes su distribución por sexo, edad, procedencia, tipo de resistencia y respuesta al tratamiento. Proponemos un manejo terapéutico para el Plasmodium vivax resistente a la cloroquina que nos fue efectivo en la totalidad de los casos, sin la necesidad de utilizar nuevas drogas

Transition of human papillomavirus type 16 and 18 transfected human foreskin keratinocytes towards immortality: activation of telomerase and allele losses at 3p, 10p, 11q and/or 18q.

This study aimed at resolving cellular genetic alterations in the process of in vitro immortalization of human keratinocytes by human papillomavirus (HPV) types 16 and 18. Four cell lines of primary human foreskin keratinocytes transfected with HPV 16 and HPV 18, respectively, were analysed during the transition from the mortal to immortal state. All cell lines showed strong telomerase activity at the immortal state, whereas no or only weak telomerase activity was detected in mortal precursor cells. This was consistent with telomere stabilization or restoration only observed in immortal cells. HPV physical state and copy number appeared constant during immortalization and HPV E6/E7 transcripts were present throughout. Immortalization was associated with clonal allele losses at 3p combined with either 11q or 18q or at 10p, dependent on the cell line. Moreover, a correlation was evident between the onset of telomerase activity and allele loss at 3p or 10p. All immortalized cells retained the capability to differentiate after growth in the presence of physiological calcium and serum. Moreover, one of the immortal cell lines displayed terminal differentiation after organotypic culturing on collagen rafts. The data suggest that (a) several pathways exist for HPV mediated immortalization that may involve genes residing at 3p, 10p, 11q and/or 18q; (b) 3p and 10p may encode genes involved in telomerase regulation; and (c) immortalization in vitro can be correlated with a spectrum of morphological changes varying from mild to severe dysplasia.

Hormone-dependent and -independent transcriptional activation by thyroid hormone receptors are mediated by different mechanisms.

Transcriptional activation by thyroid hormone (T3) receptor (T3R) generally requires the binding of its high affinity ligand. However, we reported previously that chicken T3R alpha (cTaR alpha) and human T3R beta 1 (hT3R beta 1) could activate transcription from several promoters containing T3R response elements (TREs) in a hormone-independent fashion when expressed in rat anterior pituitary GH4C1 cells. In this study we show that rat T3R alpha 1 also activates transcription without T3 in GH4C1 cells and that the oncoprotein v-erbA that is derived from cT3R alpha but does bind T3 is not a constitutive activator in these cells. Increased expression of T3R results in transcriptional activation of both native and minimal promoters, and this activation does not appear to require a defined TRE in the promoter. Because hormone-independent activity is not observed in several other cell lines, this activity may involve specific factors present in GH4C1 cells. Three mutants with single amino acid changes in a 20-amino acid region of the ligand-binding domain of cT3R alpha do not mediate hormone-independent activity. This region is highly conserved within the nuclear receptor family and has been implicated in interactions with other proteins, suggesting participation of other transcription or accessory factors in the hormone-independent activity of T3R. Two of these mutants mediate hormone-dependent transcriptional activation similar to wild-type cT3R alpha. All three mutants interact in vitro with retinoid X receptor beta similar to wild-type cT3R alpha. Our findings suggest that hormone-dependent and hormone-independent transactivation proceed by distinct mechanisms.

A 10-amino-acid sequence in the N-terminal A/B domain of thyroid hormone receptor alpha is essential for transcriptional activation and interaction with the general transcription factor TFIIB.

The effects of the thyroid hormone (3,5,3'-triiodo-L-thyronine [T3]) on gene transcription are mediated by nuclear T3 receptors (T3Rs). alpha- and beta-isoform T3Rs (T3R alpha and -beta) are expressed from different genes and are members of a superfamily of ligand-dependent transcription factors that also includes the receptors for steroid hormones, vitamin D, and retinoids. Although T3 activates transcription by mediating a conformational change in the C-terminal approximately 220-amino-acid ligand-binding domain (LBD), the fundamental mechanisms of T3R-mediated transcriptional activation remain to be determined. We found that deletion of the 50-amino-acid N-terminal A/B domain of chicken T3R alpha (cT3R alpha) decreases T3-dependent stimulation of genes regulated by native thyroid hormone response elements about 10- to 20-fold. The requirement of the A/B region for transcriptional activation was mapped to amino acids 21 to 30, which contain a cluster of five basic amino acids. The A/B region of cT3R alpha is not required for T3 binding or for DNA binding of the receptor as a heterodimer with retinoid X receptor. In vitro binding studies indicate that the N-terminal region of cT3R alpha interacts efficiently with TFIIB and that this interaction requires amino acids 21 to 30 of the A/B region. In contrast, the LBD interacts poorly with TFIIB. The region of TFIIB primarily involved in the binding of cT3R alpha includes an amphipathic alpha helix contained within residues 178 to 201. Analysis using a fusion protein containing the DNA-binding domain of GAL4 and the entire A/B region of cT3R alpha suggests that this region does not contain an intrinsic activation domain. These and other studies indicate that cT3R alpha mediates at least some of its effects through TFIIB in vivo and that the N-terminal region of DNA-bound cT3R alpha acts to recruit and/or stabilize the binding of TFIIB to the transcription complex. T3 stimulation could then result from ligand-mediated changes in the LBD which may lead to the interaction of other factors with cT3R alpha, TFIIB, and/or other components involved in the initiation of transcription.

Functional evidence for ligand-dependent dissociation of thyroid hormone and retinoic acid receptors from an inhibitory cellular factor.

The ligand-binding domains of thyroid hormone (L-triiodothyronine [T3]) receptors (T3Rs), all-trans retinoic acid (RA) receptors (RARs), and 9-cis RA receptors (RARs and RXRs) contain a series of heptad motifs thought to be important for dimeric interactions. Using a chimera containing amino acids 120 to 392 of chicken T3R alpha (cT3R alpha) positioned between the DNA-binding domain of the yeast GAL4 protein and the potent 90-amino-acid transactivating domain of the herpes simplex virus VP16 protein (GAL4-T3R-VP16), we provide functional evidence that binding of ligand releases T3Rs and RARs from an inhibitory cellular factor. GAL4-T3R-VP16 does not bind T3 and does not activate transcription from a GAL4 reporter when expressed alone but is able to activate transcription when coexpressed with unliganded T3R or RAR. This activation is reversed by T3 or RA, suggesting that these receptors compete with GAL4-T3R-VP16 for a cellular inhibitor and that ligand reverses this effect by dissociating T3R or RAR from the inhibitor. A chimera containing the entire ligand-binding domain of cT3R alpha (amino acids 120 to 408) linked to VP16 [GAL4-T3R(408)-VP16] is activated by unliganded receptor as well as by T3. In contrast, GAL4-T3R containing the amino acid 120 to 408 ligand-binding region without the VP16 domain is activated only by T3. The highly conserved ninth heptad, which is involved in heterodimerization, appears to participate in the receptor-inhibitor interaction, suggesting that the inhibitor is a related member of the receptor gene family. In striking contrast to T3R and RAR, RXR activates GAL4-T3R-VP16 only with its ligand, 9-cis RA, but unliganded RXR does not appear to be the inhibitor suggested by these studies. Further evidence that an orphan receptor may be the inhibitor comes from our finding that COUP-TF inhibits activation of GAL4-T3R-VP16 by unliganded T3R and the activation of GAL4-T3R by T3. These and other results suggest that an inhibitory factor suppresses transactivation by the T3Rs and RARs while these receptors are bound to DNA and that ligands act, in part, by inactivating or promoting dissociation of a receptor-inhibitor complex.

Detection of fetal trisomies 21 and 18 from maternal blood using triple gradient and magnetic cell sorting.

PROBLEM: The need for an inexpensive and reproducible technique for noninvasive prenatal diagnosis by fetal cell isolation from maternal blood. METHOD: For enrichment of fetal cells we used a combination of triple density gradient and magnetic sorting (MACS) of (anti-CD71) transferrin receptor antibody labeled cells followed by fluorescence in situ hybridization (FISH) with chromosome-specific DNA probes for detection of fetal aneuploidies. We identified 15 cases of fetal trisomy (five cases with a trisomy 18 and ten cases with a trisomy 21) with subsequent chromosome-specific FISH. RESULTS: We found in all of the aneuploid pregnancies that the percentage of cells with three hybridization signals did not overlap with those of normal controls independent from gestational ages and previous invasive procedures. CONCLUSIONS: Our new approach for noninvasive prenatal diagnosis has proven to be reliable in this first series.

The conserved ninth C-terminal heptad in thyroid hormone and retinoic acid receptors mediates diverse responses by affecting heterodimer but not homodimer formation.

The receptors for thyroid hormone (T3R), all-trans-retinoic acid (RAR), and 9-cis-retinoic acid (RXR) bind DNA response elements as homo- and heterodimers. The ligand-binding domains of these receptors contain nine conserved heptads proposed to play a role in dimerization. Mutant receptors with changes in the first or last hydrophobic amino acids in the highly conserved ninth heptad of chick T3R alpha [cT3R alpha(L365R) and cT3R(L372R)] and human RAR alpha (hRAR alpha) [hRAR(M377R) and hRAR(L384R)] reveal that this heptad is essential for certain heterodimeric interactions and for diverse functional activities. Without ligands, wild-type receptors form both homodimers and heterodimers, while these mutants form only homodimers. Surprisingly, the cognate ligand for each mutant enables heterodimer formation between cT3R(L365R) and RAR or RXR and between hRAR(M377R) and T3R or RXR. Both cT3R(L365R) and hRAR(M377R) mediate ligand-dependent transcriptional regulation. However, unlike the wild-type receptor, non-ligand-associated cT3R(L365R) does not suppress the basal activity of certain promoters containing thyroid hormone response elements, suggesting that this silencing effect of T3R is mediated by unliganded heterodimers of T3R and endogenous RXR or related factors. Heterodimerization is also necessary for the strong ligand-independent inhibition between T3R and RAR on a common response element, since the ninth-heptad mutants function as poor inhibitors. However, with a T3R-specific response element, hRAR(M377R) acts as a retinoic acid-dependent inhibitor of cT3R, indicating the importance of heterodimerization for this inhibition. Our studies also suggest that the ninth heptad is necessary for the dominant inhibition of wild-type T3Rs by mutant T3Rs, as has been found for the thyroid hormone-resistant syndrome in humans. Thus, the ninth heptad repeat is required for heterodimerization, suppression of basal promoter activity, and dominant negative effects of T3R and RAR. Lastly, the finding that cT3R(L365R) and hRAR(M377R) require ligands for heterodimer formation also raises the possibility that heterodimeric interactions are mediated by the ninth heptad without ligands but by a second region of these receptors with ligands.

Comparison of toxicity tests on human skin and epidermoid (A431) cells using free fatty acids as test substances.

Several in-vivo methods can be used to determine the ability of chemical compounds to induce skin irritancy. In this study we estimated in vivo the capacity of several free fatty acids to induce skin irritancy and compared the results with those found in in vitro tests. Skin irritancy induced by free fatty acids (chain lengths: C6, C7, C9, C10, C11, C13 and C18) was evaluated in humans by means of laser-Doppler flowmetry (LDF) and visual scoring (VS). Both methods demonstrated that the toxic effect of free fatty acids determined by LDF and VS increased from C6 through C11 and decreased again for C13 and C18. The cytotoxic effect of these free fatty acids on cells was measured in vitro by incubation of human epidermoid cells (A431) with these compounds. It was determined by measuring: (a) the number of dead cells by inclusion of Trypan blue (TB); and (b) the number of living cells by mitochondrial metabolism of 3-[4,5-dimethylthiazole-2-yl]-2,5 diphenyltetrazolium bromide (MTT). The LD-50 concentrations decreased from C6 through C11 in both in-vitro assays. The results of the in-vitro assays for C13 and C18 both demonstrated a discrepancy. The cytotoxic effect of the free fatty acids expressed as LD-50 values, determined after 20 min with the TB assay, was seen at higher concentrations than after incubation for 18 h (MTT assay). From the results it was concluded that C13 in particular affected skin blood flow. We also determined correlation coefficients between the in-vivo and in-vitro methods. When C13 is excluded these coefficients ranged from -0.77 to -0.92.(ABSTRACT TRUNCATED AT 250 WORDS)

CT of intracranial cryptococcosis.

CT scans of 35 patients with intracranial cryptococcal infection were reviewed retrospectively. Studies were normal in 43% of the patients. Positive findings in others included diffuse atrophy in 34%, mass lesions (cryptococcoma) in 11%, hydrocephalus in 9%, and diffuse cerebral edema in 3%. Two unusual types of cryptococcoma were encountered, namely gelatinous pseudocysts and an intraventricular cryptococcal cyst. All findings were nonspecific for CNS cryptococcosis. The results suggest that CNS cryptococcosis should be considered in all patients at risk for the disease who have these abnormal CT findings, no matter what their initial clinical presentation. In addition, MR demonstration of gelatinous pseudocysts in one patient indicates that this technique may be helpful in locating cryptococcal mass lesions not visualized on CT.

CT of intracranial cryptococcosis.

CT scans of 35 patients with intracranial cryptococcal infection were reviewed retrospectively. Studies were normal in 43% of the patients. Positive findings in others included diffuse atrophy in 34%, mass lesions (cryptococcoma) in 11%, hydrocephalus in 9%, and diffuse cerebral edema in 3%. Two unusual types of cryptococcoma were encountered, namely gelatinous pseudocysts and an intraventricular cryptococcal cyst. All findings were nonspecific for CNS cryptococcosis. The results suggest that CNS cryptococcosis should be considered in all patients at risk for the disease who have these abnormal CT findings, no matter what their initial clinical presentation. In addition, MR demonstration of gelatinous pseudocysts in one patient indicates that this technique may be helpful in locating cryptococcal mass lesions not visualized on CT.