Antibody-mediated inhibition of the growth of larvae from an insect causing cutaneous myiasis in a mammalian host

Many insects feed on blood or tissue from mammalian hosts. One potential strategy for the control of these insects is to vaccinate the host with antigens derived from the insect. The larvae of the fly Lucilia cuprina feed on ovine tissue and tissue fluids causing a cutaneous myiasis associated with considerable host morbidity and mortality. A candidate vaccine antigen, peritrophin 95, was purified from the peritrophic membrane, which lines the gut of these larvae. Serum from sheep vaccinated with peritrophin 95 inhibited growth of first-instar L. cuprina larvae that fed on this serum. Growth inhibition was probably caused by antibody-mediated blockage of the normally semipermeable peritrophic membrane and the subsequent development of an impervious layer of undefined composition on the gut lumen side of the peritrophic membrane that restricted access of nutrients to the larvae. The amino acid sequence of peritrophin 95 was determined by cloning the DNA complementary to its mRNA. The deduced amino acid sequence codes for a secreted protein containing a distinct Cys-rich domain of 317 amino acids followed by a mucin-like domain of 139 amino acids. The Cys-rich domain may be involved in binding chitin. This report describes a novel immunological strategy for the potential control of L. cuprina larvae that may have general application to the control of other insect pests.

Activation of Jak/STAT proteins involved in signal transduction pathway mediated by receptor for interleukin 2 in malignant T lymphocytes derived from cutaneous anaplastic large T-cell lymphoma and Sezary syndrome.

Signaling through the interleukin 2 receptor (IL-2R) involves phosphorylation of several proteins including Jak3, STAT5, and, in preactivated cells, STAT3. In the present study, we examined the functional status of the IL-2R-associated Jak/STAT pathway in malignant T lymphocytes from advanced skin-based lymphomas: anaplastic large T-cell lymphoma (ALCL) and Sezary syndrome (SzS). Proliferation of three ALCL cell lines (PB-1, 2A, and 2B) was partially inhibited by rapamycin, a blocker of some of the signals mediated by IL-2R, but not by cyclosporin A, FK-506, and prednisone, which suppress signals mediated by the T-cell receptor. All the cell lines expressed on their surface the high-affinity IL-2R (alpha, beta, and gamma c chains). They showed basal, constitutive phosphorylation, and coassociation of Jak3, STAT5, and STAT3. Weak basal phosphorylation of IL-2R gamma c was also detected. In regard to SzS, peripheral blood mononuclear cells from 10 of 14 patients showed basal phosphorylation of Jak3, accompanied by phosphorylation of STAT5 in 9 patients, and STAT3 in 4 patients. However, in vitro overnight culture of SzS cells without exogenous cytokines resulted in markedly decreased Jak3 and STAT5 phosphorylation, which could be reversed by stimulation with IL-2. This indicates that the basal phosphorylation of Jak3 and STAT5 in freshly isolated SzS cells is induced rather than constitutive. The basal activation of the Jak/STAT pathway involved in IL-2R signal transduction in ALCL and SzS cells reported here suggests that this pathway may play a role in the pathogenesis of cutaneous T-cell lymphomas, although the mechanism (induced versus constitutive) may vary between different lymphoma types.

Low dimensionality of supraspinally induced force fields

Recent experiments using electrical and N-methyl-d-aspartate microstimulation of the spinal cord gray matter and cutaneous stimulation of the hindlimb of spinalized frogs have provided evidence for a modular organization of the frog’s spinal cord circuitry. A “module” is a functional unit in the spinal cord circuitry that generates a specific motor output by imposing a specific pattern of muscle activation. The output of a module can be characterized as a force field: the collection of the isometric forces generated at the ankle over different locations in the leg’s workspace. Different modules can be combined independently so that their force fields linearly sum. The goal of this study was to ascertain whether the force fields generated by the activation of supraspinal structures could result from combinations of a small number of modules. We recorded a set of force fields generated by the electrical stimulation of the vestibular nerve in seven frogs, and we performed a principal component analysis to study the dimensionality of this set. We found that 94% of the total variation of the data is explained by the first five principal components, a result that indicates that the dimensionality of the set of fields evoked by vestibular stimulation is low. This result is compatible with the hypothesis that vestibular fields are generated by combinations of a small number of spinal modules.

Chymase cleavage of stem cell factor yields a bioactive, soluble product

Stem cell factor (SCF) is produced by stromal cells as a membrane-bound molecule, which may be proteolytically cleaved at a site close to the membrane to produce a soluble bioactive form. The proteases producing this cleavage are unknown. In this study, we demonstrate that human mast cell chymase, a chymotrypsin-like protease, cleaves SCF at a novel site. Cleavage is at the peptide bond between Phe-158 and Met-159, which are encoded by exon 6 of the SCF gene. This cleavage results in a soluble bioactive product that is 7 amino acids shorter at the C terminus than previously identified soluble SCF. This research shows the identification of a physiologically relevant enzyme that specifically cleaves SCF. Because mast cells express the KIT protein, the receptor for SCF, and respond to SCF by proliferation and degranulation, this observation identifies a possible feedback loop in which chymase released from mast cell secretory granules may solubilize SCF bound to the membrane of surrounding stromal cells. The liberated soluble SCF may in turn stimulate mast cell proliferation and differentiated functions; this loop could contribute to abnormal accumulations of mast cells in the skin and hyperpigmentation at sites of chronic cutaneous inflammation.

Functional characterization of the C—C chemokine-like molecules encoded by molluscum contagiosum virus types 1 and 2

Many viruses have evolved mechanisms for evading the host immune system by synthesizing proteins that interfere with the normal immune response. The poxviruses are among the most accomplished at deceiving their hosts’ immune systems. The nucleotide sequence of the genome of the human cutaneous poxvirus, molluscum contagiosum virus (MCV) type 1, was recently reported to contain a region that resembles a human chemokine. We have cloned and expressed the chemokine-like genes from MCV type 1 and the closely related MCV type 2 to determine a potential role for these proteins in the viral life cycle. In monocyte chemotaxis assays, the viral proteins have no chemotactic activity but both viral proteins block the chemotactic response to the human chemokine, macrophage inflammatory protein (MIP)-1α. Like MIP-1α, both viral proteins also inhibit the growth of human hematopoietic progenitor cells, but the viral proteins are more potent in this activity than the human chemokine. These viral chemokines antagonize the chemotactic activity of human chemokines and have an inhibitory effect on human hematopoietic progenitor cells. We hypothesize that the inhibition of chemotaxis is an immune evasion function of these proteins during molluscum contagiosum virus infection. The significance of hematopoietic progenitor cell inhibition in viral pathogenesis is uncertain.

Insulin-like growth factor I is a growth-promoting factor for Leishmania promastigotes and amastigotes

Leishmaniases are diseases caused by protozoa of the genus Leishmania that affect more than 20 million people in the world. The initial phase of the infection is fundamental for either the progression or control of the disease. The Leishmania parasites are injected in the skin as promastigotes and then, after been phagocytized by the host macrophages, rapidly transform into amastigotes. In this phase different nonspecific cellular and humoral elements participate. We have shown previously that insulin-like growth factor (IGF)-I that is constitutively present in the skin induces growth of Leishmania promastigotes. In the present paper we show further evidence for the importance of this factor: (i) IGF-I also can induce a growth response in Leishmania (Leishmania) mexicana amastigotes; (ii) IGF-I binds specifically to a putative single-site receptor on both promastigotes and amastigotes; (iii) IGF-I induces a rapid tyrosine phosphorylation of parasite proteins with different molecular mass in promastigotes and amastigotes of L. (L.) mexicana; and, finally, (iv) the cutaneous lesion in the mice when challenged by IGF-I-preactivated Leishmania (Viannia) panamensis is increased significantly because of inflammatory process and growth of parasites. We thus suggest that IGF-I is another important host factor participating in the Leishmania–host interplay in the early stage during the establishment of the infection and presumably also in the later stages.

CTACK, a skin-associated chemokine that preferentially attracts skin-homing memory T cells

In contrast to naive lymphocytes, memory/effector lymphocytes can access nonlymphoid effector sites and display restricted, often tissue-selective, migration behavior. The cutaneous lymphocyte-associated antigen (CLA) defines a subset of circulating memory T cells that selectively localize in cutaneous sites mediated in part by the interaction of CLA with its vascular ligand E-selectin. Here, we report the identification and characterization of a CC chemokine, cutaneous T cell-attracting chemokine (CTACK). Both human and mouse CTACK are detected only in skin by Southern and Northern blot analyses. Specifically, CTACK message is found in the mouse epidermis and in human keratinocytes, and anti-CTACK mAbs predominantly stain the epithelium. Finally, CTACK selectively attracts CLA+ memory T cells. Taken together, these results suggest an important role for CTACK in recruitment of CLA+ T cells to cutaneous sites. CTACK is predominantly expressed in the skin and selectively attracts a tissue-specific subpopulation of memory lymphocytes.

The inhibition of antigen-presenting activity of dendritic cells resulting from UV irradiation of murine skin is restored by in vitro photorepair of cyclobutane pyrimidine dimers

Exposing skin to UVB (280–320 nm) radiation suppresses contact hypersensitivity by a mechanism that involves an alteration in the activity of cutaneous antigen-presenting cells (APC). UV-induced DNA damage appears to be an important molecular trigger for this effect. The specific target cells in the skin that sustain DNA damage relevant to the immunosuppressive effect have yet to be identified. We tested the hypothesis that UV-induced DNA damage in the cutaneous APC was responsible for their impaired ability to present antigen after in vivo UV irradiation. Cutaneous APC were collected from the draining lymph nodes of UVB-irradiated, hapten-sensitized mice and incubated in vitro with liposomes containing a photolyase (Photosomes; Applied Genetics, Freeport, NY), which, upon absorption of photoreactivating light, splits UV-induced cyclobutane pyrimidine dimers. Photosome treatment followed by photoreactivating light reduced the number of dimer-containing APC, restored the in vivo antigen-presenting activity of the draining lymph node cells, and blocked the induction of suppressor T cells. Neither Photosomes nor photoreactivating light alone, nor photoreactivating light given before Photosomes, restored APC activity, and Photosome treatment did not reverse the impairment of APC function when isopsoralen plus UVA (320–400 nm) radiation was used instead of UVB. These controls indicate that the restoration of APC function matched the requirements of Photosome-mediated DNA repair for dimers and post-treatment photoreactivating light. These results provide compelling evidence that it is UV-induced DNA damage in cutaneous APC that leads to reduced immune function.

Selective increase in specific alternative splice variants of tyrosinase in murine melanomas: A projected basis for immunotherapy

Melanomas tend to become less pigmented in the course of malignant progression. Thus, as proliferation increases, the tumors are decreasingly characterized by the tissue-specific phenotype of normally differentiated melanocytes. To learn whether the decline in melanization is associated with a shift from constitutive to alternative splicing of some pigment gene pre-mRNAs, melanomas were collected from Tyr-SV40E transgenic mice of the standard C57BL/6 strain. The mRNAs of the tyrosinase gene, which has a key role in melanogenesis, were analyzed by quantitative reverse transcriptase–PCR in 34 samples from 16 cutaneous tumors and 9 metastases. The cutaneous tumors included some cases with distinct melanotic and amelanotic zones, which were separately analyzed. All tyrosinase transcripts found in the melanomas were also found in normal skin melanocytes. However, the Δ1b and Δ1d alternatively spliced transcripts, due to deletions within the first exon, were specifically augmented in most of the tumors over their very low levels in skin; the exceptions were some all-amelanotic tumors in which no tyrosinase transcripts were detected. The level of Δ1b rose as high as 11.3% of total tyrosinase mRNAs as compared with 0.6% in skin; Δ1d reached 4.0% as compared with 0.8% in skin. Expression of these splice variants was highest in the melanotic components of zonal primary tumors, relatively lower in their amelanotic components, and still lower in all-amelanotic primary tumors and amelanotic metastases. The increase in Δ1b and Δ1d transcripts may be predicted to increase the levels of unusual peptides, which could have antigenic potential in the tumors, especially in the relatively early phases of malignancy. Analyses of the alternative transcripts of other pigment genes may identify additional candidate antigens, ultimately enabling melanoma cells in all phases of the disease to be represented as a basis for immune intervention.

Down-regulation of transcripts for Na channel α-SNS in spinal sensory neurons following axotomy

Spinal sensory (dorsal root ganglion; DRG) neurons display slowly inactivating, tetrodotoxin-resistant (TTX-R), and rapidly inactivating, TTX-sensitive (TTX-S) Na currents. Attenuation of the TTX-R Na current and enhancement of TTX-S Na current have been demonstrated in cutaneous afferent DRG neurons in the adult rat after axotomy and may underlie abnormal bursting. We show here that steady-state levels of transcripts encoding the α-SNS subunit, which is associated with a slowly inactivating, TTX-R current when expressed in oocytes, are reduced significantly 5 days following axotomy of DRG neurons, and continue to be expressed at reduced levels, even after 210 days. Steady-state levels of α-III transcripts, which are present at low levels in control DRG neurons, show a pattern of transiently increased expression. In situ hybridization using α-SNS- and α-III-specific riboprobes showed a decreased signal for α-SNS, and an increased signal for α-III, in both large and small DRG neurons following axotomy. Reduced levels of α-SNS may explain the selective loss of slowly inactivating, TTX-R current. The abnormal electrophysiological properties of DRG neurons following axonal injury thus appear to reflect a switch in Na channel gene expression.

Identification of sample-specific sequences in mammalian cDNA and genomic DNA by the novel ligation-mediated subtraction (Limes)

The representational difference analysis (RDA) and other subtraction techniques are used to enrich sample-specific sequences by elimination of ubiquitous sequences existing in both the sample of interest (tester) and the subtraction partner (driver). While applying the RDA to genomic DNA of cutaneous lymphoma cells in order to identify tumor relevant alterations, we predominantly isolated repetitive sequences and artificial repeat-mediated fusion products of otherwise independent PCR fragments (PCR hybrids). Since these products severely interfered with the isolation of tester-specific fragments, we developed a considerably more robust and efficient approach, termed ligation-mediated subtraction (Limes). In first applications of Limes, genomic sequences and/or transcripts of genes involved in the regulation of transcription, such as transforming growth factor β stimulated clone 22 related gene (TSC-22R), cell death and cytokine production (caspase-1) or antigen presentation (HLA class II sequences), were found to be completely absent in a cutaneous lymphoma line. On the assumption that mutations in tumor-relevant genes can affect their transcription pattern, a protocol was developed and successfully applied that allows the identification of such sequences. Due to these results, Limes may substitute/supplement other subtraction/comparison techniques such as RDA or DNA microarray techniques in a variety of different research fields.

Inherited susceptibility to uterine leiomyomas and renal cell cancer

Herein we report the clinical, histopathological, and molecular features of a cancer syndrome with predisposition to uterine leiomyomas and papillary renal cell carcinoma. The studied kindred included 11 family members with uterine leiomyomas and two with uterine leiomyosarcoma. Seven individuals had a history of cutaneous nodules, two of which were confirmed to be cutaneous leiomyomatosis. The four kidney cancer cases occurred in young (33- to 48-year-old) females and displayed a unique natural history. All these kidney cancers displayed a distinct papillary histology and presented as unilateral solitary lesions that had metastasized at the time of diagnosis. Genetic-marker analysis mapped the predisposition gene to chromosome 1q. Losses of the normal chromosome 1q were observed in tumors that had occurred in the kindred, including a uterine leiomyoma. Moreover, the observed histological features were used as a tool to diagnose a second kindred displaying the phenotype. We have shown that predisposition to uterine leiomyomas and papillary renal cell cancer can be inherited dominantly through the hereditary leiomyomatosis and renal cell cancer (HLRCC) gene. The HLRCC gene maps to chromosome 1q and is likely to be a tumor suppressor. Clinical, histopathological, and molecular tools are now available for accurate detection and diagnosis of this cancer syndrome.

DEG/ENaC ion channels involved in sensory transduction are modulated by cold temperature

Several DEG/ENaC cation channel subunits are expressed in the tongue and in cutaneous sensory neurons, where they are postulated to function as receptors for salt and sour taste and for touch. Because these tissues are exposed to large temperature variations, we examined how temperature affects DEG/ENaC channel function. We found that cold temperature markedly increased the constitutively active Na+ currents generated by epithelial Na+ channels (ENaC). Half-maximal stimulation occurred at 25°C. Cold temperature did not induce current from other DEG/ENaC family members (BNC1, ASIC, and DRASIC). However, when these channels were activated by acid, cold temperature potentiated the currents by slowing the rate of desensitization. Potentiation was abolished by a “Deg” mutation that alters channel gating. Temperature changes in the physiologic range had prominent effects on current in cells heterologously expressing acid-gated DEG/ENaC channels, as well as in dorsal root ganglion sensory neurons. The finding that cold temperature modulates DEG/ENaC channel function may provide a molecular explanation for the widely recognized ability of temperature to modify taste sensation and mechanosensation.

Forebrain mechanisms of nociception and pain: Analysis through imaging

Pain is a unified experience composed of interacting discriminative, affective-motivational, and cognitive components, each of which is mediated and modulated through forebrain mechanisms acting at spinal, brainstem, and cerebral levels. The size of the human forebrain in relation to the spinal cord gives anatomical emphasis to forebrain control over nociceptive processing. Human forebrain pathology can cause pain without the activation of nociceptors. Functional imaging of the normal human brain with positron emission tomography (PET) shows synaptically induced increases in regional cerebral blood flow (rCBF) in several regions specifically during pain. We have examined the variables of gender, type of noxious stimulus, and the origin of nociceptive input as potential determinants of the pattern and intensity of rCBF responses. The structures most consistently activated across genders and during contact heat pain, cold pain, cutaneous laser pain or intramuscular pain were the contralateral insula and anterior cingulate cortex, the bilateral thalamus and premotor cortex, and the cerebellar vermis. These regions are commonly activated in PET studies of pain conducted by other investigators, and the intensity of the brain rCBF response correlates parametrically with perceived pain intensity. To complement the human studies, we developed an animal model for investigating stimulus-induced rCBF responses in the rat. In accord with behavioral measures and the results of human PET, there is a progressive and selective activation of somatosensory and limbic system structures in the brain and brainstem following the subcutaneous injection of formalin. The animal model and human PET studies should be mutually reinforcing and thus facilitate progress in understanding forebrain mechanisms of normal and pathological pain.