Melanotic Nodules Research Articles

Plasmatocyte-spreading peptide (PSP) plays a central role in insect cellular immune defenses against bacterial infection

Insect hemocytes (blood cells) are a central part of the insect's cellular response to bacterial pathogens, and these specialist cells can both recognize and engulf bacteria. During this process, hemocytes undergo poorly characterized changes in adhesiveness. Previously, a peptide termed plasmatocyte-spreading peptide (PSP), which induces the adhesion and spreading of plasmatocytes on foreign surfaces, has been identified in lepidopteran insects. Here, we investigate the function of this peptide in the moth Manduca sexta using RNA interference (RNAi) to prevent expression of the precursor protein proPSP. We show that infection with the insect-specific bacterial pathogen Photorhabdus luminescens and non-pathogenic Escherichia coli induces proPSP mRNA transcription in the insect fat body but not in hemocytes; subsequently, proPSP protein can be detected in cell-free hemolymph. We used RNAi to silence this upregulation of proPSP and found that the knock-down insects succumbed faster to infection with P. luminescens, but not E. coli. RNAi-treated insects infected with E. coli showed a reduction in the number of circulating hemocytes and higher bacterial growth in hemolymph as well as a reduction in overall cellular immune function compared with infected controls. Interestingly, RNAi-mediated depletion of proPSP adversely affected the formation of melanotic nodules but had no additional effect on other cellular responses when insects were infected with P. luminescens, indicating that this pathogen employs mechanisms that suppress key cellular immune functions in M. sexta. Our results provide evidence for the central role of PSP in M. sexta cellular defenses against bacterial infections.

A single locus from the entomopathogenic bacteriumPhotorhabdus luminescensinhibits activatedManduca sextaphenoloxidase

Insect blood (hemolymph) contains prophenoloxidase, a proenzyme that is activated to protective phenoloxidase when the insect is damaged or challenged with microorganisms. The Gram-negative bacterium Photorhabdus luminescens kills the lepidopteron insect Manduca sexta by using a variety of toxins. We screened P. luminescens and Photorhabdus asymbiotica cosmid libraries in an Escherichia coli host against previously activated M. sexta hemolymph phenoloxidase and identified three overlapping cosmid clones from P. luminescens and five from P. asymbiotica that suppressed the activity of the enzyme both in vitro and in vivo. Genome alignments of cosmid end sequences from both species confirmed that they contained orthologous loci. We examined one of the cosmids from P. luminescens in detail: it induced the formation of significantly fewer melanotic nodules, proliferated faster within the insect host and was significantly more virulent towards fifth-stage larvae than E. coli control bacteria. Insertional mutagenesis of this cosmid yielded 11 transposon mutants that were no longer inhibitory. All of these were insertions into a single 5.5-kb locus, which contained three ORFs and was homologous to the maltodextrin phosphorylase locus of E. coli. The implications of this novel inhibitory factor of insect phenoloxidase for Photorhabdus virulence are discussed.

Volcano Ulcers in Stomach – an Unusual Presentation of Metastatic Non Pigmented Melanoma

Purpose: Malignant melanoma is one of the most common malignancies to metastasize to the gastrointestinal (GI) tract. Metastases to GI tract can present at time of primary diagnosis or decades later as first sign of recurrence. Metastatic melanoma to GI tract is found during diagnostic workup in 1%-4% of patients with cutaneous primary and up to 60% of melanoma patients in autopsy. Methods: Our patient is a 45 year old white male with history of primary non-pigmented urinary bladder melanoma who presented with complaint of dyspepsia for 2 months. Patient had received full course of chemotherapy with Tamodar for 5 months with good response. He denied any nausea, vomiting, dysphagia or bleeding. He had no primary skin lesion found on extensive detailed physical exam. Whole body PET scan showed markedly thickened stomach walls with multiple omental lesions in the perigastric region. Upper endoscopy revealed two large, non-pigmented ulcerating masses in body of stomach on the greater curvature. These masses had “volcano appearance” with heaped up edges and central crater. Multiple biopsies were taken which were consistent with non pigmented metastatic melanoma confirmed by immunohistochemical stains. Patient was subsequently started on carboplatin and has had mild improvement in symptoms. Results: Conclusion: Gastrointestinal invasion by melanoma is a rare condition and is often associated with invasion of other visceral organs. The endoscopic classification of the gastric metastases comprises three main types: (a) melanotic nodules, often ulcerated at the tip; most common (b) submucosal mass, melanotic or not, elevated and ulcerated at apex; typical aspect of “bull's eye” lesion and (c) mass lesions with varying incidence of necrosis and melanosis. Gastric metastases may appear even as a simple ulcer. Majority of gastric metastases are reported to occur in body and fundus, most often on the greater curvature. Most frequent sites of melanoma metastases include small bowel, stomach, colon, and anorectum in decreasing order. Symptoms may include abdominal pain, dysphagia, small bowel obstruction and GI bleed. Diagnosis requires careful inspection of mucosa and biopsy with special immunohistochemical stains. Management may include surgical resection, chemotherapy, immunotherapy, observation, or enrollment in clinical trials. Surgery seems to be of limited value and should be performed in carefully selected patients and in patients with complications. Prognosis is poor, with median survival time in patients presenting with GI invasion being less than 1 year. Endoscopic “Volcano ulcers” in the stomach have been reported in many secondary neoplasms of the stomach and their presence should always raise suspicion for an underlying malignancy.

Developmental modulation of immunity: Changes within the feeding period of the fifth larval stage in the defence reactions of Manduca sexta to infection by Photorhabdus

In insect pathogen interactions, host developmental stage is among several factors that influence the induction of immune responses. Here, we show that the effectiveness of immune reactions to a pathogen can vary markedly within a single larval stage. Pre-wandering fifth-stage (day 5) larvae of the model lepidopteran insect Manduca sexta succumb faster to infection by the insect pathogenic bacterium Photorhabdus luminescens than newly ecdysed fifth-stage (day 0) caterpillars. The decrease in insect survival of the older larvae is associated with a reduction in both humoral and cellular defence reactions compared to less developed larvae. We present evidence that older fifth-stage larvae are less able to over-transcribe microbial pattern recognition protein and antibacterial effector genes in the fat body and hemocytes. Additionally, older larvae show reduced levels of phenoloxidase (PO) activity in the cell-free hemolymph plasma as well as a dramatic decrease in the number of circulating hemocytes, reduced ability to phagocytose bacteria and fewer melanotic nodules in the infected tissues. The decline in overall immune function of older fifth-stage larvae is reflected by higher bacterial growth in the hemolymph and increased colonization of Photorhabdus on the basal surface of the insect gut. We suggest that developmentally programmed variation in immune competence may have important implications for studies of ecological immunity.

An antibiotic produced by an insect-pathogenic bacterium suppresses host defenses through phenoloxidase inhibition

Photorhabdus is a virulent pathogen that kills its insect host by overcoming immune responses. The bacterium also secretes a range of antibiotics to suppress the growth of other invading microorganisms. Here we show that Photorhabdus produces a small-molecule antibiotic (E)-1,3-dihydroxy-2-(isopropyl)-5-(2-phenylethenyl)benzene (ST) that also acts as an inhibitor of phenoloxidase (PO) in the insect host Manduca sexta. The Photorhabdus gene stlA encodes an enzyme that produces cinnamic acid, a key precursor for production of ST, and a mutation in stlA results in loss of ST production and PO inhibitory activity, which are both restored by genetic complementation of the mutant and also by supplying cinnamic acid. ST is produced both in vitro and in vivo in sufficient quantities to account for PO inhibition and is the only detectable solvent-extractable inhibitor. A Photorhabdus stlA- mutant is significantly less virulent, proliferates slower within the host, and provokes the formation of significantly more melanotic nodules than wild-type bacteria. Virulence of the stlA- mutant is also rescued by supplying cinnamic acid. The proximate cause of the virulence effect, however, is the inhibition of PO, because the effect of the stlA- mutation on virulence is abolished in insects in which PO has been knocked down by RNA interference (RNAi). Thus, ST has a dual function both as a PO inhibitor to counter host immune reactions and also as an antibiotic to exclude microbial competitors from the insect cadaver.

The immunoglobulin family protein Hemolin mediates cellular immune responses to bacteria in the insect Manduca sexta

Bacterial recognition in the lepidopteran insect, Manduca sexta, is mediated by pattern recognition proteins including Hemolin, Peptidoglycan recognition protein (PGRP) and Immulectin-2. These proteins bind to molecular patterns present on the surface of bacteria and trigger a protective response involving humoral and cellular reactions. Cellular mechanisms mediated by haemocytes include phagocytosis, encapsulation, and the formation of melanotic nodules. Here, we show that a non-pathogenic strain of Escherichia coli induces mRNA transcription and protein expression of Hemolin and PGRP but not Immulectin-2 in Manduca haemocytes. This upregulation can be effectively prevented (knocked-down) using RNA interference (RNAi) following injection of double-stranded (ds) RNA. Knock-down of Hemolin significantly decreased the ability of insects to clear E. coli from the haemolymph and caused a reduction in the number of free haemocytes. RNAi of Hemolin reduced the ability of haemocytes to engulf bacteria through phagocytosis and to form melanotic nodules in vivo. Importantly, washed haemocytes taken from RNAi-treated insects showed reduced ability to form microaggregates around bacteria in vitro. This shows that the immune function affected by RNAi knock-down of Hemolin is intrinsic to the haemocytes. In contrast, RNAi of PGRP had no effect on any of these cellular immune functions. These results demonstrate the vital role of Hemolin in Manduca cellular immune responses.

Nuclear factor-kappa B pathways in Drosophila

The nuclear factor kappa B (NF-kappaB) pathways in Drosophila are multi-component pathways, as in vertebrates, that regulate the expression of many genes responsible for the formation of dorsal-ventral polarity in the early embryo, the innate immune response to infection with Gram- negative and positive bacteria and fungi, the cellular immune response and hematopoiesis. Overactivation of the fly pathway can result in developmental defects, overproliferation of hemocytes and the formation of melanotic tumors or nodules. The extracellular events leading to the maturation of the ligand for initiation of the Drosophila NF-kappaB pathway is not conserved between flies and vertebrates, but the Toll receptor and downstream events are remarkably similar. NF-kappaB proteins have been identified in mollusks, and arthropods such as horseshoe crabs and beetles, indicating that this pathway has been established more than 500 million years ago. The fly NF-kappaB pathways are less complex than those in vertebrates, with the involvement of fewer proteins, but they are, nonetheless, just as important as their vertebrate counterparts for the life of the fly.

Melanotic Mutants in Drosophila: Pathways and Phenotypes

Mutations in >30 genes that regulate different pathways and developmental processes are reported to cause a melanotic phenotype in larvae. The observed melanotic masses were generally linked to the hemocyte-mediated immune response. To investigate whether all black masses are associated with the cellular immune response, we characterized melanotic masses from mutants in 14 genes. We found that the melanotic masses can be subdivided into melanotic nodules engaging the hemocyte-mediated encapsulation and into melanizations that are not encapsulated by hemocytes. With rare exception, the encapsulation is carried out by lamellocytes. Encapsulated nodules are found in the hemocoel or in association with the lymph gland, while melanizations are located in the gut, salivary gland, and tracheae. In cactus mutants we found an additional kind of melanized mass containing various tissues. The development of these tissue agglomerates is dependent on the function of the dorsal gene. Our results show that the phenotype of each mutant not only reflects its connection to a particular genetic pathway but also points to the tissue-specific role of the individual gene.

The Effects of Eicosanoid Biosynthesis Inhibitors On Prophenoloxidase Activation, Phagocytosis and Cell Spreading in Galleria mellonella

The invertebrate immune system produces melanotic nodules in response to bacterial infections and this has previously been shown to be mediated by eicosanoids. Nodulation occurs in two phases: the first involves hemocyte degranulation and activation of the prophenoloxidase cascade; the second involves formation of a cellular capsule by attachment and spreading of hemocytes. We demonstrate that inhibitors of eicosanoid biosynthesis affect both of these phases of nodulation in Galleria mellonella. The phospholipase A 2 inhibitor, dexamethasone, as well as the cyclooxygenase inhibitor, indomethacin, significantly inhibit phagocytosis in vitro and prophenoloxidase activation in vivo. The inhibitory effects of dexamethasone were abolished by the addition of exogenous arachidonic acid. Furthermore, 5,8,11,14- eicosatetraynoic acid, dexamethasone and indomethacin inhibit hemocyte spreading in vitro. The findings support the idea that eicosanoid derivatives mediate both phases of the nodulation response and are consistent with previous studies which attribute roles for eicosanoids in other species as modulators of cell activity.

Inducing melanotic nodules within the fat body of the last instar larvae of Epilachnà varivestis (Coleoptera) by azadirachtin

Injection of minute amounts of azadirachtin causes formation of numerous small (20- to 30-μm diameter) nodules in the fat body of last instar larvae of the Mexican bean beetle, Epilachna varivestis. About 1 day after injection, some fat body cells start degenerating by necrosis. The first visible sign is a leakage of the membranes as detected by a tracer substance (colloidal lanthanum nitrate). The remnants of the distributed fat cells are pinocytosed and possibly phagocytosed by other fat cells. Subsequently, such fat cells develop large heterolysosomes. After the degeneration of these cells, the heterolysosomes become free within the intercellular spaces of the fat body and clot with remmants of other cells by forming large aggregations which become black during condensation. During this process, hemocytes form a multilayered coat around the aggregations, which then stop growing. For several reasons it is presumed that the formation of these melanotic nodules is correlated with the interference of azadirachtin with premetamorphic events in the larvae.

Note on the experimental use of rabies vaccine for melanomatosis.

In May 1940 a 32 year old woman with a malignant melanoma 3 cm. in diameter and situated in the pretibial region had the lesion surgically excised on the mixed tumor service of the Memorial Hospital. An elective groin dissection six weeks later revealed no deposits of metastatic melanoma in regional nodes. In September 1942 three cutaneous melanotic nodules were excised from the same leg and thigh. In September 1943, after a dog bite, the patient received 14 injections of rabies vaccine. In April 1948 two additional melanotic nodules were removed from the skin on the ipsolateral side. At this writing, two years later, there had been no further evidence of recurrence. The long latent period of 5½ years intervening between the appearance of multiple metastases was so extraordinarily unusual that it excited considerable speculation among various members of the clinic staff concerning the cause of this inhibition. In a