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Insight into brown spider and loxoscelism

Authors:

Abstract

Loxosceles is a genus of cosmopolitan spiders comprising several species, and popularly knownas brown spiders or brown recluses. Brown spider bites can cause dermonecrotic lesions andsystemic reactions known as loxoscelism. Systemic effects are less common but may be severe oreven fatal in some patients. Systemic manifestations include intravascular hemolysis, disseminatedintravascular coagulation and acute renal failure. A rapid diagnosis and an understanding of thevenom’s molecular activity are crucial for satisfactory treatment. Mechanisms by which venoms exerttheir deleterious effects are under investigation, and searches are underway for diagnosticenvenomation assays. Molecular biology is being used to produce quantities of several of the mostimportant venom molecules and has contributed to the study and understanding of their mechanismsof action.
152
ISJ 2: 152-158, 2005 ISSN 1824-307X
Review
Insights into brown spider and loxoscelism
MH Appel
1
, R Bertoni da Silveira
1,3
, W Gremski
1,2
, SS Veiga
1
1
Department of Cell Biology, Federal University of Paraná, Jardim das Américas, Curitiba, Paraná , Brazil
2
Catholic University of Paraná, Health and Biological Sciences Institute, Curitiba, Paraná Brazil
3
Department of Biochemistry, Federal University of São Paulo, São Paulo, Brazil
Accepted December 27, 2005
Abstract
Loxosceles is a genus of cosmopolitan spiders comprising several species, and popularly known
as brown spiders or brown recluses. Brown spider bites can cause dermonecrotic lesions and
systemic reactions known as loxoscelism. Systemic effects are less common but may be severe or
even fatal in some patients. Systemic manifestations include intravascular hemolysis, disseminated
intravascular coagulation and acute renal failure. A rapid diagnosis and an understanding of the
venom’s molecular activity are crucial for satisfactory treatment. Mechanisms by which venoms exert
their deleterious effects are under investigation, and searches are underway for diagnostic
envenomation assays. Molecular biology is being used to produce quantities of several of the most
important venom molecules and has contributed to the study and understanding of their mechanisms
of action.
Key words: brown spider; loxoscelism; venom; recombinant toxins; dermonecrosis
Introduction
More than 40,000 spider species exist, with
probably 100,000 to be described, but only 3 taxa are
recognized as dangerous, namely Theridiidae,
Loxoscelidae and Ctenidae. Moreover, only the
genera Atrax, Lactrodectus and Loxosceles are
associated with human deaths (Escoubas et al., 2000;
Rash and Hodgson, 2002). Early European tales
during the Middle Ages linked injuries or illness to
spider bites (Schienle et al., 2005). For example the
tarantula bite was associated with a disease
(tarantism) for which the cure was a frenetic dancing
for 3-4 days. This energetic dance, called tarantella, is
now a typical Italian dance (Isbister, 2004). Today, as
a consequence of mistaken diagnoses of spider bites,
scientists are looking for methods to characterize and
identify spider bites and their manifestations as well
as to better understand the biological and molecular
Corresponding Author:
Silvio S. Veiga
Department of Cell Biology, Federal University of Paraná,
Jardim das Américas,81531-990, Curitiba, Paraná,
Brazil
E-mail: veigass@ufpr.br
mechanisms of envenomation.
The genus Loxosceles (variously known as the
brown spider, brown recluse, fiddleback, or gaucho
spiders) is important in these studies because of its
commonness in and around human dwellings. Their
bite is characterized by dermonecrosis and systemic
effects known as loxoscelism (Hogan et al., 2004).
The first case of documented loxoscelism
occurred in 1879 in Tennessee. However, consistent
data traced back about 50 years ago and were
collected in Chile, then other observations were made
in Brazil followed by the United States. These reports
linked brown spider bite with necrotic skin lesions
(Macchiavello, 1947; Atkin et al., 1958; Sams et al.,
2001). Spiders’ habits have caused a close
association with humans, and the number of bites is
increasing and has become a public health problem in
Brazil, Chile and the United States (da Silva et al.,
2004). Most bites occur during sleep or dressing, and
women are bitten more often than men. Thighs, trunk,
hands and arms are more often bitten (Hogan et al.,
2004).
Loxosceles spiders
Loxosceles spiders are known as violin
(fiddleback) spiders due to a characteristic violin
153
shape on their cephalothorax (Futrell, 1992). They are
also known as brown spiders because their colour
varies from a pale (L. laeta) to a dark brown (L.
gaucho). Loxosceles body length ranges from 8 to 15
mm with legs measuring from 8 to 30 mm (da Silva et
al., 2004). They are sedentary and nocturnal (Andrade
et al., 1999) with a lifetime of 3 7 years (Andrade et
al., 2000). Brown spiders have three pairs of eyes (an
important characteristic useful to identify the genus)
(Vetter and Visscher, 1998). They build irregular,
cottony webs (Futrell, 1992) and normally prefer dead
scavenged prey rather than live preys (Sandidge,
2003). They can survive months without food or water
and withstand temperatures ranging from 8 °C to 43
°C. They are not aggressive and prefer dark dry
places (Futrell, 1992; Málaque et al., 2002; Vetter and
Barger, 2002; da Silva et al., 2004). The sexes
produce venom with differences in volume, toxicity
and compounds proportion (Oliveira et. al., 1999).
Comparative analysis of sex and species in L. laeta
and L. intermedia venom showed some biological
activities (complement-dependent hemolysis and
dermonecrosis) more prominent in venom from female
spiders, especially from L. laeta (Oliveira et al., 2005).
Epidemiology
Loxosceles spiders can be found distributed all
over the world. In North America, the most important
species are L. reclusa, L. deserta, L arizona, L.
rufences (United States and Mexico) and L. laeta
(Canada) (Sams et al., 2001; Vetter and Bush,
2002a). Europe, Africa, Middle East, some parts of
Asia, Israel, and Australia are hosts to some
Loxosceles species (Futrell, 1992; Borkkan et al.,
1995; Young and Pincus, 2001; Nicholson and
Graudins, 2003).
In Brazil, seven species have been described but
three are the most implicated in human bites L.
intermedia, L. gaucho and L. laeta (Sezerino et al.,
1998). From 1990 to 1993, the Brazilian Ministry of
Health received 17.781 reports of spiders’ bites, of
which 36 % were due to Loxosceles (Sezerino et al.,
1998). In the metropolitan area of Curitiba, in the state
of Parana (southern Brazil) about 3.000 brown spider
bites are reported annually (Málaque et al., 2002). In
a retrospective study in Florianopolis, in the state of
Santa Catarina, Brazil, 487 suspected cases of brown
spider bites were found, 267 of which fulfilled the
criteria for inclusion in the study (Sezerino et al.,
1998). In 359 cases of loxoscelism between January
1985 and December 1996 at Butantan Intitute, São
Paulo, Brazil, 14 % of patients captured the spiders so
that 28 were classified as L. gaucho, 5 as L. laeta and
18 as non-classified Loxosceles (Málaque et al.,
2002). More bites occur in warmer months (Schenone,
1996). In Curitiba, from 1998 to 2001 the incidence of
Loxosceles bites was 1.4 cases per 1,000 habitants.
Of these, 23 % were in the thigh, 16.7 % in the trunk,
14 % in the arm and 13 % in the lower leg. Only 1 %
of cases were severe (Health Secretary, Curitiba,
Parana, Brazil, 2002).
Pathophysiology of Loxoscelism
Dermonecrosis is the hallmark of loxoscelism
(Fig. 1). Histopathology and clinical data are obtained
from biopsies of human patients after brown spider
bites. Rabbit skin artificially injected with Loxosceles
venom is used for more controlled investigation since
this animal model reproduces human skin lesions that
follow envenomation (Ospedal et al., 2002). Systemic
effects, such as renal failure, are less common and
are usually reproduced in mouse (Luciano et al.,
2004). Observation of human skin biopsies showed an
inflammatory infiltrate, thrombosis, hemorrhage,
dermatitis, erythema, induration of affected area and
liquefactive necrosis of the epidermis and dermis
consistent with pyoderma grangrenosum (Futrell,
1992; Yannias and Winkelmann, 1992). Symptoms in
an experimental study in rabbits showed that after 4 h
oedema, hemorrhage, degeneration of blood vessel
walls, plasma exudation, thrombosis, neutrophil
accumulation in and around blood vessels with an
intensive diapedesis, a diffuse collection of
inflammatory cells (polymorphonuclear leucocytes) in
the dermis, and subcutaneous muscular oedema all
occur. Over the following hours and up to 5 days after
envenomation, the changes progressed to a massive
neutrophil infiltration into the dermis and even into
subcutaneous muscle tissue, destruction of blood
vessels, thrombosis, hemorrhage, myonecrosis, and
coagulative necrosis on the 5
th
day (Ospedal et al.,
2002). Neutrophil participation and the inflammatory
response seem to be dependent on an endothelial cell
agonist effect triggered by the venom that leads to an
indirect and dysregulated neutrophil activation
involved in dermonecrosis (Patel, 1994).
Envenomation of rabbit skin with L. reclusa venom
after 14 days results in a mixed inflammatory cell
infiltrate, coagulative tissue necrosis, vasculitis and a
dense band of neutrophils bordering the zone of
necrosis (Elston et al., 2000). L. intermedia venom
damaged vessel endothelia, as shown by vessel
instability, endothelium cell vacuolization in biopsies of
rabbit skin (Veiga et al., 2001a; Zanetti et.al., 2002). In
vitro experiments on rabbit aorta endothelium cell
cultures showed cytotoxicity of L. intermedia venom
associated with loss of cell adhesion to the culture
substrate and the shedding of proteoglycans from the
extracellular matrix and cell surface into the medium
(Veiga et al., 2001a). In human umbilical vein
endothelial cell (HUVEC) cultures treated with L.
reclusa venom, agonist activity ensued, inducing
endothelial cell expression of E-selectin and the
release of interleukin (IL)-8 and granulocyte
macrophage colony-stimulating factor, resulting in
dysregulated inflammatory response (Patel et al.,
1994). HUVEC exposed to L. deserta venom produced
IL-8, growth-related oncogene-α and monocyte
chemoattractant protein-1 via an NF-κB- dependent
pathway (Desai et al., 1999; Gomez et al., 1999). L.
deserta venom induces the expression of vascular
endothelial growth factor (VEGF) in human
keratinocytes, suggesting that keratinocyte-derived
VEGF may contribute to vasodilatation, oedema and
erythema in brown spider envenomation (Desai et al.,
2000). Primary cultures of keratinocytes exposed to
100 ng/ml of L. gaucho venom release tumour
necrosis factor (TNF)-α into the medium after 6 h
(Málaque et al., 1999).
Mice injected with L. reclusa venom developed
local hemorrhage after 6 h accompanied by blistering
of the ear skin (Sunderkötter et al., 2001).
154
Fig. 1 Cellular and molecular aspects of brown spider and loxoscelism. (A) Loxosceles intermedia (brown spider)
male. (B) L. intermedia (brown spider) female. (C) SDS-PAGE 3-20 % venom profile stained by Coomassie blue
dye. (D) Dermonecrotic lesion on rabbit skin after 24 h post-L. intermedia venom (10 µg) exposure. Arrowhead
indicates the site of venom injection with characteristic black and white eschar named marble plate. Black arrow
points an erythema surrounding the lesion and white arrow shows the gravitational spreading of lesion (a hallmark
of dermonecrotic loxoscelism). (E) Microscopical view of dermonecrotic lesion showing inflammatory leukocytes
accumulated in the connective tissue (arrowhead) and disorganization of collagen fiber and oedema (black arrow)
(magnification 400X). The inset shows inflammatory cells of the infiltrate represented by neutrophils (white arrow)
(magnification 1.000X).
Histopathology showed a vasculitis reaction 2 h
after exposure. The microscopical analysis of some
mouse organs injected with different doses of L.
intermedia venom revealed remarkable kidney
alterations. Acute tubular necrosis accompanied by
deposition of eosinophilic material inside the proximal
and distal renal tubules was seen in several nephrons
(Tambourgi et.al., 1998). Mouse kidneys, treated with
L. intermedia venom showed hyalinisation and
erythrocytes in the Bowman’s space, glomerular
collapse, tubular epithelial cell cytotoxicity and
deposition of eosinophilic material within the tubular
lumen (Luciano et al., 2004). Confocal microscopy
observations of double staining immunofluorescence
against type IV collagen or laminin and L. intermedia
venom showed that toxins deposit and bind along the
tubular and glomerular basement membrane of mice
kidneys. Ultrastructural observations showed
glomerular epithelial and endothelial cell cytotoxicity,
the collapse and destruction of glomerular basement
membrane and tubular epithelial cell degeneration.
The basement membrane is a target for brown spider
venom, as shown administrating L. intermedia venom
to murine tumor Engelbreth-Holm-Swarm (EHS),
which is rich in basement membrane molecules. L.
intermedia venom degraded and fragmented the
basement membrane (Veiga et.al., 2000a). Venom
29
-
44
-
12
-
116
-
205
-
kD
a
male
female
A
B
C
D
E
1cm
1cm
1cm
155
displays hydrolytic activity for entactin and heparan
sulphate proteoglycan, two important constituents of
basement membranes, while having no apparent
activity on purified type IV collagen and laminin (Veiga
et al., 2000a, 2001a,b).
In the bone marrow and peripheral blood cells, L.
intermedia initially causes a decrease in the number
of nucleated red cells, bone-marrow depression of
megakariocytes with thrombocytopenia in peripheral
blood and decrease of platelet count (da Silva et al.,
2003). Neutropenia in peripheral blood and low
neutrophil counts were observed as consequence of
bone-marrow depletion, which may reflect an
extensive neutrophil influx to the tissues. Eosinophils
are apparently unaffected.
Brown spider venom toxins
L. intermedia and L. laeta have different protein
patterns of glycosylation and the same is between
sexes of the same species (Oliveira et al., 2005).
Hemolytic and dermonecrotic activities have been
described for L. similes venom. Sphingomyelinase D
molecules, with molecular mass ranging from 30 to 35
kDa and having hemolytic, necrotic and platelet
aggregation activity were found in L. reclusa, L.
rufescens, L. gaucho, L. laeta and L. intermedia
venoms (Futrell 1992; Barbaro et al., 1994; Mota and
Barbaro, 1995; Tambourgi et al., 1995; Barbaro et al.,
1996a,b, 1997). Three sphingomyelinase D isoforms
were purified from L. boneti venom (Lb1, Lb2 and
Lb3). Only Lb1 and Lb2 had dermonecrotic activity
(Ramos-Cerrillo et al., 2004). An alkaline phosphatase
was described in L. reclusa venom (Futrell, 1992).
Hyaluronidase (32.5 kDa) was found in L. refescens
and L. reclusa (Futrell, 1992; Young and Pincus,
2001). L. deserta, L. gaucho, L. intermedia, L. laeta
and L. reclusa venoms contained an enzyme of
similar molecular size (44 kDa), which digested
hyaluronic acid (Barbaro et al., 2005). A 5’-
ribonucleotide phosphohydrolase was found in L.
reclusa venom (Futrell 1992). Loxnecrogin A (31.4
kDa) and Loxnecrogin B (31.6 kDa) with necrotic
activity on rabbit skin were found in L. gaucho venom
(Cunha et al., 2003). L. intermedia has a range of
proteases described in its venom: Loxolysin A (20-28
kDa) with fibronectinolytic and fibrinogenolytic activity;
Loxolysin B (32-35 kDa) with gelatinolytic activity
(Feitosa et al., 1998); a serin protease (85 kDa) with
gelatinolytic activity (Veiga et.al., 2000b) and
proteases able to hydrolyse entactin, heparan
sulphate proteoglican and basement membrane
(Veiga et al., 2000b, 2001a,b). L. rufescens also has a
broad molecular range of caseinolytic, gelatinolytic
and fibrogenolytic metalloproteases (Young and
Pincus, 2001). To test whether proteases in L.
intermedia venom were due to natural constitution and
not a digest fluid contamination, da Silveira et al.,
(2002) compared the proteolytic activity of the venom
obtained directly from venom glands with that
obtained by electroshock. Both protein profiles
showed very similar electrophoretic and enzymatic
characteristics.
At present, a new generation of molecules
developed through cloning techniques is under study.
L. intermedia LiD1 recombinant protein (31.4 kDa) is a
sphingomyelinase D family molecule without
dermonecrotic activity but with antigenic activity
(Kalapothakis et al., 2002). L. laeta recombinant
protein (33 kDa) is a sphingomyelinase isoform able to
degrade sphingomyelin (Pedrosa et al., 2002). This
recombinant protein induced complement
susceptibility, release of glycophorins and had
dermonecrotic activity. L. intermedia recombinant
protein (LiRecDT, 34 kDa) has dermonecrotic activity
and was able to directly induce nephrotoxicity in mice
(Chaim et al., 2005). L. laeta recombinant
phospholipase D generated lysophosphatidic acid and
was hemolytic (Lee and Lynch, 2005).
Clinical features, diagnosis and treatment of
brown spider bites
Diagnosis of loxoscelism is rarely based on spider
identification and therefore clinical features,
epidemiological and historical findings must be well
known (Wright et al., 1997; Vetter, 1999; Málaque et
al., 2002). Lesion recovery improves once the patient
is treated. However, brown recluse bites have been
misdiagnosed in North America because they
occurred in regions of non-endemicity (Vetter, 1999;
Nishioka, 2001; Vetter and Barger, 2002; Vetter and
Bush, 2002a,b; Vetter et al., 2003). A typical necrotic
skin lesion begins soon after the spider bites the
victim, followed by gravitational spreading (da Silva et
al., 2004). The bite is painless, hence the patient is
often unaware that he has been bitten (Futrell, 1992),
and the delay between the bite and when the victim
pursues help makes the treatment less effective. From
mild to severe pain begins 2-8 h after the bite. At the
bite a small puncture wound may appear, associated
with transient erythema with itching and swelling and
mild to severe tenderness (Futrell, 1992; da Silva et
al., 2004). Blebs or blisters appear (12-24 h), may
become hemorrhagic, and surrounded by a halo of
ischemic tissue. In the following days, necrotized
lesions become a dull blue-violet, the area of the
gravitational spread turns blue, and the size of the
blue area increases. Within three to seven days an
eschar may form, after which the lesion hardens. The
eschar may drop off leaving an ulcer that may require
a skin graft (Schenone, 1996; Sezerino et al., 1998;
Málaque et al., 2002; da Silva et al., 2004).
Success of therapy depends upon a correct and
rapid diagnosis, the volume of the venom injected, and
the patient susceptibility to the venom (Futrell, 1992;
da Silva et al., 2004). Phentolamine, heparin, topical
nitro-glycerine, cyproheptadine and hyperbaric
oxygenation have been used for therapy, but the
efficacy of these therapies is inconclusive and their
use is not recommended (Futrell, 1992; Wendell,
2003; da Silva et al., 2004). The established therapy is
dapsone, acetylsalicylic acid (aspirin), antibiotics
(erythromycin and cephalosporin), ice and elevation,
avoidance of strenuous activity and heat and, when
necessary, surgery. Early surgical excision has not
been shown to be effective and often delays healing
(Futrell, 1992; Merigian and Blaho, 1996; Goddard,
1998, Monteiro et al., 2002; da Silva et al., 2004).
Serum anti-Loxosceles venom is used only in severe
cases and effectiveness is doubtful especially against
local manifestation. Systemic envenomation studies in
156
animals and humans have demonstrated that
antivenom neutralizes the deleterious effects of the
venom and reduces paediatric mortality (Isbister et al.,
2003). Effectiveness of antivenom to prevent
dermonecrotic lesions seems to be time dependent
and usually patient looks for medical help 4 h after the
bite when lesions is already established (Ospedal et
al., 2002; Nicholson and Graudins, 2003). Some local
and systemic noxious activities of the venom are
attributed to proteolytic toxins that degrade fibrinogen,
fibronectin, entactin and heparan sulphate
proteoglycan and disrupt basement membrane
structures, thereby causing local hemorrhage,
gravitational spreading, disseminated intravascular
coagulation and renal failure (Feitosa, et al., 1998;
Veiga et al., 1999, 2000b, 2001a,b; Luciano et al.,
2004; Chaim et al., 2005).
Biotechnological products from brown spider
venom
Recently developed technologies are being used
to produce biotechnological products from Loxosceles
venom. ARACHnase (Hemostasis Diagnosis
International Co., Denver, CO, USA), normal plasma
containing L. reclusa venom, mimics a lupus
anticoagulant and may provide a positive control for
anticoagulant testing (McGlasson et al., 1993). An
antiserum against venoms of L. gaucho, Phoneutria
nigriventer, Tityus serrulatus, and Tityus bahiensis
that reacts with L. intermedia and L. laeta toxins is
produced by The Butantan Institute, São Paulo, Brazil.
The CCPI (Production Center of Immunobiologic
Products, Parana, Brazil) has also produced
antiserum using L. intermedia venom that is able to
neutralize some activities of Loxosceles venom. L.
laeta antiserum is produced by the National Institute
of Health (Peru) (da Silva et al., 2004). These antisera
have all been used as bioproducts for serum therapy
(Roodt et al., 2002; Barbaro et al., 1994; 1996a;
Health Secretary, Curitiba, Parana, Brazil). Guilherme
et al. (2001) produced monoclonal antibodies
recognising L. gaucho venom toxins, which were able
to neutralize the dermonecrotic effect and lethal
activities of this species venom but not those of
heterologous venoms.
Monoclonal and polyclonal antibodies are not
only powerful tools for neutralizing the effects of
venom; they are also useful for research. They can be
used to purify toxins from venom by affinity
chromatography. They can be used on location of
specific toxins on cell and tissue treated with venom
toxins. Immunofluorescence techniques such as
confocal microscopy and flow cytometry are modern
techniques based on antibody specific binding.
In contrast to the collection of snake venom,
spiders provide very little venom, which limits the
ability to study spider venom toxins. Protein cloning
techniques are helping to solve this problem. After
cloning it is possible to have milligrams of the same
protein thereby improving the quality of research work
and allowing more controlled experimental studies.
Today several spider venom recombinant proteins are
under investigation most of which are in the
sphingomyelinase protein family (Kalapothakis et al.,
2002; Pedrosa et al., 2002; Chaim et al., 2005).
Future perspectives
Toxins from Loxosceles spiders are a group of
proteins with a great range of different activities. Each
toxin may be used to investigate molecular and
cellular effects of venom. Also each of these proteins
is a putative molecular model for drug design and to
develop knowledge on some effects not yet fully
understood such as the inflammatory reaction of
dermonecrosis and platelet aggregation.
Acknowledgements
This work was supported by CNPq, CAPES,
Fundação Araucária and Secretaria de Estado de
Ciência, Tecnologia e Ensino Superior do Parana,
Brazil.
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... Monoclonal antibodies neutralizing phospholipases-D and metalloprotease activities were previously produced by our group [21,41,42]. In this sense, it is important to produce a mAb capable of neutralizing the hyaluronidase activity of Loxosceles venom, since this toxin is responsible for increasing venom diffusion and spreading of the lesion [6,9,10]. In conclusion, this study indicated that rMEPLox quantity and solubility are increased when it is produced under an optimized protocol. ...
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Loxoscelism is the most dangerous araneism form in Brazil and antivenom therapy is the recommended treatment. Antivenom is produced by horse immunization with Loxosceles spider venom, which is toxic for the producer animal. Moreover, due to the high amount of venom required for horse hyperimmunization, new strategies for antigens obtention have been proposed. In this sense, our research group has previously produced a non-toxic recombinant multiepitopic protein derived from Loxosceles toxins (rMEPLox). rMEPLox was a successful immunogen, being able to induce the production of neutralizing antibodies, which could be used in the Loxoscelism treatment. However, rMEPLox obtention procedure requires optimization, as its production needs to be scaled up to suit antivenom manufacture. Therefore, an effective protocol development for rMEPlox production would be advantageous. To achieve this objective, we evaluated the influence of different cultivation conditions for rMEPLox optimum expression. The optimum conditions to obtain large amounts of rMEPlox were defined as the use of C43(DE3)pLysS as a host strain, 2xTY medium, 0.6 mM IPTG, biomass pre induction of OD600nm = 0.4 and incubation at 30 °C for 16 h. Following the optimized protocol, 39.84 mg/L of soluble rMEPLox was obtained and tested as immunogen. The results show that the obtained rMEPLox preserved the previously described immunogenicity, and it was able to generate antibodies that recognize different epitopes of the main Loxosceles venom toxins, which makes it a promising candidate for the antivenom production for loxoscelism treatment.
... Systemic effects are less common (about 10% of cases), but may be severe or even fatal in some patients; they include nausea, vomiting, chills, fever, myalgia, generalized urticaria or purpuric rash, morbilliform rash, hemolytic anemia, disseminated intravascular coagulation, acute renal failure, shock, coma, and death [7]. There is no diagnostic test available to quickly help the physician make a diagnostic or therapeutic decision, but a rapid diagnosis is crucial for satisfactory treatment [7], [12]. In the present paper, a case, which we presumed to be due to the bite of Loxosceles rufescens, is evaluated in the light of literature [13], [14], [15], [16], [17], [18], [19], [20], [21]. ...
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BACKGROUND: The spiders of the Loxosceles genus, commonly denoted as “brown spiders” or “Mediterranean recluse” or “brown recluse,” belong to the spider family Sicariidae, suborder Labidognatha, order Araneida, class Arachnida, and phylum Arthropoda. This spider is widespread in Africa and South/Central America, but it is also distributed in North America, in the West Indies, in the Mediterranean Europe, and in China. CASE REPORT: Here, we report the case of a severe dermonecrotic loxoscelism identified in Southern Italy, probably due to the bite of Loxosceles rufescens. The patient was a women admitted at hospital ER because of a little skin erythema that evolved toward a severe necrosis and ulceration within 20 days. After clinical and laboratory data excluded other local and systemic diseases, she was treated with a systemic and local therapy using corticosteroids and antibiotics with the diagnosis of loxoscelism. The healing from the local skin lesion occurred within 2 months, but the local pain, weakness, and discomfort lasted for a long time. CONCLUSION: It is the fisrt time that a possible case of systemic loxoscelism with skin generalized urticaria is reported in Italy.
... The venom of Loxosceles spp. is composed of numerous protein molecules with toxic and/or enzymatic activity [2, 3,[8][9][10][11], such as phospholipases D, metalloproteases, serine proteases, hyaluronidases, allergens, serine protease inhibitors, and peptides classified as cystine knot inhibitors [9,[12][13][14][15][16]. Studies have shown that phospholipases D (PLDs) are the most abundant toxins able to elicit a cascade of adverse pharmacological events such as inflammation [13,17] dermonecrosis [11,13,[18][19][20][21], platelet aggregation [21][22][23], hemolysis [13,23,24], and nephrotoxicity [25,26], among others. ...
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Human accidents with spiders of the genus Loxosceles are an important health problem affecting thousands of people worldwide. Patients evolve to severe local injuries and, in many cases, to systemic disturbances as acute renal failure, in which cases antivenoms are considered to be the most effective treatment. However, for antivenom production, the extraction of the venom used in the immunization process is laborious and the yield is very low. Thus, many groups have been exploring the use of recombinant Loxosceles toxins, particularly phospholipases D (PLDs), to produce the antivenom. Nonetheless, some important venom activities are not neutralized by anti-PLD antibodies. Astacin-like metalloproteases (ALMPs) are the second most expressed toxin acting on the extracellular matrix, indicating the importance of its inclusion in the antigen’s formulation to provide a better antivenom. Here we show the construction of a hybrid recombinant immunogen, called LgRec1ALP1, composed of hydrophilic regions of the PLD and the ALMP toxins from Loxosceles gaucho. Although the LgRec1ALP1 was expressed as inclusion bodies, it resulted in good yields and it was effective to produce neutralizing antibodies in mice. The antiserum neutralized fibrinogenolytic, platelet aggregation and dermonecrotic activities elicited by L. gaucho, L. laeta, and L. intermedia venoms, indicating that the hybrid recombinant antigen may be a valuable source for the production of protective antibodies against Loxosceles ssp. venoms. In addition, the hybrid recombinant toxin approach may enrich and expand the alternative antigens for antisera production for other venoms.
... SMase D from Loxosceles species venoms are denominated "dermonecrotic toxins" due to their ability to cause dermonecrosis in vivo [Appel et al., 2005;Chaim et al., 2011a;Gremski et al., 2014]. CpSMaseD causes in vivo dermonecrosis in rabbits after 3 hours of exposure. ...
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Sphingomyelinases D have only been identified in arachnid venoms, Corynebacteria, Arcanobacterium, Photobacterium and in the fungi Aspergillus and Coccidioides. The arachnid and bacterial enzymes share very low sequence identity and do not contain the HKD sequence motif characteristic of the phospholipase D superfamily, however, molecular modeling and circular dichroism of SMases D from L. intermedia and C. pseudotuberculosis indicate similar folds. The phospholipase, hemolytic and necrotic activities and mice vessel permeabilities were compared and both enzymes possess the ability to hydrolyze phospholipids and also promote similar pathological reactions in the host suggesting the existence of a common underlying mechanism in tissue disruption. This article is protected by copyright. All rights reserved.
... Phospholipases D interact with cell membranes and other elements in tissue triggering alterations which involve the complement system and activation of neutrophils. More important is the response of host cells to the presence of a complement mediating foreign substance [6]. Painful bites may be noticed immediately and the offending spider may be seen; often, a diagnosis is made in retrospect from a characteristic history and physical examination. ...
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The spider bites are quite frequent and often resolve quickly without leaving outcomes; only some species are capable of causing necrotic and systematic lesions in humans. Among them, we should mention the genus Loxosceles . The venom released from the spider bite of Loxosceles species is composed of proteins, enzymes, and nonenzymatic polypeptides. The phospholipase D family was identified as the active component of the venom. This family of enzymes is responsible for the local and systemic effects observed in loxoscelism. Phospholipases D interact with cell membranes triggering alterations which involve the complement system and activation of neutrophils and they cause the dermonecrotic skin lesions and systemic effects. We describe a fatal case of acute intoxication caused by a spider bite probably belonging to the species Loxosceles . The initial lesion was localized to a finger of a hand. Clinical course was worsening with deep necrotic lesions on limb, shock, hemolysis, acute kidney failure, and disseminated intravascular coagulation. All therapies were ineffective. This is the first fatal case described in Europe.
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Loxoscelismo é o termo utilizado para os sintomas clínicos desencadeados após a picada de aranhas do gênero Loxosceles. As manifestações clínicas incluem necrose da pele com espalhamento gravitacional e complicações sistêmicas. O veneno loxoscélico contém várias enzimas, como por exemplo, fosfolipases-D, serinoproteases, metaloproteases e hialuronidases. As hialuronidases ao degradar glicosaminoglicanos de tecido conjuntivo potencializaria a ação de outros componentes do veneno facilitando a penetração e distribuição dos mesmos a diferentes tecidos. Este trabalho teve como objetivo a clonagem, expressão e caracterização bioquímica e biológica de uma isoforma de hialuronidase do veneno de L. intermedia (aranha-marrom). Através de uma biblioteca de cDNA da glândula de veneno, uma isoforma de hialuronidase (1200 pb) foi clonada e denominada Dietrich´s Hyaluronidase (DHAase). Após subclonagem da hialuronidase madura em vetor de expressão pET-14b, a construção resultante foi transformada em Escherichia coli BL21(DE3)pLysS e AD494(DE3). Fora testada a expressão em volume de 50 mL em diferentes concentrações de indutor IPTG (0,05-0,4 mM) e diferentes temperaturas (30º, 22º e 16ºC). Para obtenção da solubilidade da enzima recombinante, a sequência da hialuronidase madura foi também clonada em vetor de expressão pET-SUMO e expressa em E. coli SHuffle a 30ºC sob as mesmas condições de indutor descritas anteriormente. Em um maior volume de cultura (1L) nenhuma das cepas foi eficiente em expressar a DHAase solúvel. Sendo assim a alternativa escolhida para conseguir a hialuronidase recombinante de forma solúvel e ativa foi a técnica de redobramento in vitro. Vários tampões foram testados e a atividade só foi obtida com um tampão contendo glutationas, albumina bovina (BSA) e L-arginina. Depois do redobramento, DHAase foi capaz de degradar ácido hialurônico (HA) e condroitim-4-sulfato (C4S) em ensaios de cinética e zimografia. Por meio de análises de imunodetecção foi visto que anticorpos policlonais produzidos com a DHAase desnaturada e com o veneno reagiram de forma cruzada. Através de experimentos in vivo de dermonecrose em pele de coelho, foi demonstrado que DHAase aumentou a área de necrose produzida por uma isoforma de fosfolipase-D recombinante (toxina dermonecrótica) do veneno de L. intermedia (LiRecDT1). Dados macroscópicos e histológicos suportam a hipótese de que a hialuronidase é um "fator de espalhamento" do veneno de L. intermedia. Adicionalmente, estudando modelos in vitro de células endoteliais de aorta de coelho (RAEC) e células de melanoma murino (B16F1 e B16F10) onde essas células foram expostas por até 24 h com DHAase, foi visto que a hialuronidase recombinante não induziu alteração da morfologia ou desadesão das RAECs. Por outro lado ela alterou a proliferação e a morfologia das células de melanoma murino, sendo que a linhagem mais metastática (B16F10) teve os efeitos mais pronunciados sob a ação de DHAase. A viabilidade celular de B16F10 diminui significativamente com a exposição da hialuronidase recombinante por 16 h. Dietrich´s Hyaluronidase foi uma ferramenta útil para um estudo pioneiro no loxoscelismo e se mostrou eficiente em estudar o efeito do catabolismo de HA em linhagens cancerosas de melanoma, embora estudos adicionais se façam necessários. Em conclusão, o presente trabalho tornou evidente a possível utilização de DHAase como uma ferramenta de estudo em processos patológicos relacionados ao ácido hialurônico.
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El objetivo del estudio fue describir el comportamiento espacial y temporal de los casos de loxoscelismo registrados en la sala virtual de situación de salud del Centro Nacional de Epidemiología, Prevención y Control de Enfermedades (CDC-Perú) en el periodo 2009 – 2018. Se recolectó información correspondiente a las variables grupo etáreo, sexo, año y región geográfica en el que se reportaron las mordeduras. Con la información recopilada se creó una base de datos en Microsoft Excel la que fue resumida mediante estadística descriptiva (frecuencias absolutas y relativas). Se observó una tendencia creciente en el reporte de accidentes loxoscélicos, alcanzando el máximo número en el año 2016. El grupo etario más afectado fue el de 30 a 59 años (4 108 casos) seguido del de 18 a 29 años (2 014 casos).. En general, las mujeres fueron las más afectadas (5 265 casos), pero en la población infantil, los casos se reportaron con mayor frecuencia en los varones. Lima fue la región con mayor número de reportes (2 958 casos), no obstante, la región con mayor tasa de presentación fue Arequipa con 150,9 casos por cada 100 000 habitantes. El estudio mostró que en regiones y temporadas de mayor temperatura los casos de loxoscelismo aumentaban. Además, se observó que los accidentes loxoscélicos pueden ocurrir a cualquier edad por diversos factores de exposición, por lo que sería importante implementar campañas de prevención orientadas a disminuir el riesgo de exposición en las diferentes etapas de la vida de las personas.
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Spider venoms are complex mixtures of proteins, peptides and small organic and inorganic molecules. Among the proteins, phospholipases D (PLDs) present the major portion, and till now they are the most studied enzymes in spider venom. These PLDs have been divided into two classes, I and II, based on their primary and tertiary structure. Currently, crystal structures of both classes of these enzymes are available in the Protein Data Bank (PDB). Their three-dimensional structure is composed of eight α-helices and eight β-strands forming the ubiquitous fold called triosephosphate isomerase (TIM) barrel. These enzymes use general acid-base catalysis to hydrolyzes their substrate. In this review, we have described the structural features, structure-based mechanisms of catalysis, maturation, and inhibition of these enzymes using the synthetic inhibitor.
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Introduction: Loxoscelism represents a major public health problem for which there are no standard thera-peutic interventions. Objective: To review available scientific evidence on management of Loxoscelism Method: Systematic review of clinical studies. The search included múltiple databases (Medline, Lilacs, Embase, Web of Sciences, Cinahl, Pre-Cinahl, Paperfirst, Proceedingsfirst, Dissertations and Theses, Toxline, Cochrane Library), handsearch of references, and contact with experts. Results: Three clinical triáis of poor methodological quality were identified from 5,207 references found. One trial (n = 31), concluded that the use of dapsone was associated with fewer local complications than surgical treatment. A second study (n = 46), concluded that the use of dapsone was superior to clorfenamine for skin lesions. A third study (n = 95) concluded that there was no differences be-tween the use of oral dapsone, antivenom against anti-Loxosceles reclusa or a combination of both. Conclusions: There is insufficient evidence based on good quality studies to recommend treatment guidelines for individuáis with skin or visceral loxoscelism.
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Brown recluse spider (Loxosceles reclusa) venom induces severe dermonecrotic lesions. The mechanism for this is unknown but presents an interesting paradox: necrosis is completely dependent on the victim's neutrophils, yet neutrophils are not activated by the venom. We show Loxosceles venom is a potent, but disjointed, endothelial cell agonist. It weakly induced E-selectin expression, but not intercellular adhesion molecule-1 or IL-6 expression, yet significantly stimulated release of IL-8 and large amounts of GM-CSF by 4 h. In contrast, TNF strongly induced all of these, except for GM-CSF. PMN bound to E-selectin on venom-activated endothelial cells, apparently via counterreceptors different from those that bind E-selectin on TNF alpha-activated monolayers. Notably, PMN bound venom-activated monolayers only at intercellular junctions, did not polarize, and completely failed to migrate beneath the monolayer. Despite this, bound PMN demonstrated increased intracellular Ca2+ levels and secreted primary and secondary granule markers. The latter event was suppressed by sulfones used to treat envenomation. We have defined a new endothelial cell agonist, Loxosceles venom, that differentially stimulates the inflammatory response of endothelial cells. This, in turn, leads to a dysregulated PMN response where adhesion and degranulation are completely dissociated from shape change and transmigration.
Article
1. The biological, immunochemical and biochemical properties of venoms from Loxosceles gaucho, L. laeta and L. intermedia were studied. 2. Venom from Loxosceles sp causes typical dermonecrotic lesion in bitten patients and rabbits but not in mice. 3. The dermonecrotic and lethal activities of the L. gaucho venom were located in its higher mol. wt components (33.000–35.000). 4. The higher molecular component of the venom is the most immunogenic component in both patients and rabbits. 5. Analysis by SDS-PAGE showed the existence of many common components in the three venoms but no individual antigen was found. 6. Antiserum to each one of these venoms was able to neutralize the dermonecrotic and lethal activities of all three venoms. 7. Venom from L. gaucho has a significant adjuvant effect that was associated with its higher mol. wt components. 8. The amino terminal sequence of 35 residues of L. gaucho active component was determined. A high level of identity (60%) and similarity (86%) was found between the L. gaucho toxic protein and a fragment of L. reclusa venom toxic protein suggesting that they are homologous proteins.
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Bites from the brown recluse spider and other Loxosceles arachnids result in dermonecrotic skin lesions. Neutrophils (PMN) are essential to the development of Loxosceles-induced skin lesions, but paradoxically, in vitro PMN activation is inhibited by direct exposure to Loxosceles venom. Neutrophil activation occurs in response to a myriad of soluble mediators that include members of both the and chemokine families. Because arachnid envenomation results in the exposure of several different cell types to venom, we investigated venom-induced expression of and chemokines in both endothelial cells (human umbilical vein; HUVEC) and epithelial cells (A549 pneumocytes). Chemokine-specific capture enzyme immunoassays (EIA) were used to measure Loxosceles deserta venom-induced chemokines: interleukin-8 (IL-8), growth-related oncogene-alpha (GRO-), and chemokines: monocyte chemoattractant protein-1 (MCP-1), and regulated on activation, normal T cell expressed and secreted (RANTES) in cell-free conditioned media from HUVEC and A549 cell monolayers. Exposure of HUVECs (8 h) to Loxosceles venom resulted in the production of IL-8 (5.2 1.30 ng/ml), MCP-1 (1.44 0.11 ng/ml) and GRO- (1.97 0.15 ng/ml) in a dose and time-dependent manner. Exposure of A549 cell monolayers to venom resulted in IL-8 (7.74 0.30 ng/ml), and MCP-1 (2.61 0.31 ng/ml), but neither GRO- nor RANTES accumulated during an 8-hour incubation period. Chemokines accumulated in a venom dose and time-dependent manner. Neither cell type secreted RANTES in response to Loxosceles venom. These data indicate that Loxosceles spider venom is a potent inducer of and chemokines in both endothelial and epithelial cell types. Based on the established roles of IL-8, MCP-1, and GRO-, in inflammation, these observations have relevance to the pathophysiology of Loxosceles-Induced dermonecrosis.
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There are several groups of medically important araneomorph and mygalomorph spiders responsible for serious systemic envenomation. These include spiders from the genus Latrodectus (family Theridiidae), Phoneutria (family Ctenidae) and the subfamily Atracinae (genera Atrax and Hadronyche); The venom of these spiders contains potent neurotoxins that cause excessive neurotransmitter release via vesicle exocytosis or modulation of voltage-gated sodium channels.-In addition, spiders of the genus Loxosceles (family Loxoscelidae) are responsible for significant local reactions resulting in necrotic cutaneous lesions. This results from sphingomyelinase D activity and possibly other compounds. A number of antivenoms are currently available to treat envenomation resulting from the bite of these spiders. Particularly efficacious antivenoms are available for Latrodectus and Atrax/Hadronyche species, with extensive cross-reactivity within each genera. In the case of Latrodectus antivenoms this is of considerable importance in countries where antivenom is unavailable or where certain antivenoms are associated with an unacceptably high risk,of adverse reactions. Moreover, Latrodectus and Atrax antivenoms appear, to be effective in the treatment of envenomation by closely related Steatoda spiders (family Theridiidae) or the unrelated spider Missulena bradleyi (family Actinopodidae), respectively., The effectiveness of Loxosceles antivenom in the treatment of the necrotic arachnidism resulting from the bite of recluse spiders is less clear mainly due to late presentation of victims. Antivenom is also available for Phoneutria envenomation but is reserved only for severe cases.
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Neutralization of dermonecrotic and lethal activities and differences among the principal toxic proteins (32–35 kDa) of medically important Loxosceles spider venoms in Brazil (Loxosceles gaucho, Loxosceles laeta and Loxosceles intermedia) were studied using monoclonal antibodies (MAbs) produced against the dermonecrotic component (35 kDa) of L. gaucho venom. MAb titers were 512,000 to homologous venom, between 2000 and 64,000 for L. intermedia venom and between 1000 and 64,000 for L. laeta venom. By Western blotting, MAbs could recognize mainly the 35 kDa protein of L. gaucho venom and with less intensity the 35 kDa protein of L. intermedia venom. These MAbs also recognized weakly or did not recognize the 32 kDa component of L. laeta venom. Only MoALg1 showed high affinity for L. gaucho venom and neutralized in vivo 90–97% of the dermonecrotic activity, besides delaying the lethality induced by homologous venom. MoALg1 maintained its capacity to neutralize the dermonecrotic activity, even when administered (i.v.) 6 h after envenoming (i.d.). All MAbs obtained failed to neutralize the toxic activities of the heterologous venoms.These results suggest that different epitopes are present in the protein responsible for the dermonecrotic activity of Loxosceles venoms, and confirm the participation of other venom components during the local reaction process. This study also confirms the importance of antibodies for neutralization of dermonecrotic activity, even when administered some hours after envenoming, and emphasizes the differences of composition and toxicity of medically important Loxosceles venoms. These findings must be considered in order to improve loxoscelism immunotherapy.
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The bite of the arthropod Loxosceles is known to cause subdermal hemorrhage, dermal-epidermal separation, inflammatory infiltrates, as well as occlusion and necrosis of small arterioles. We report a case of a brown recluse spider bite that presented as a chronic painful skin plaque, with the unusual histologic findings of a cutaneous and deep subcutaneous hyalinizing panniculitis and myonecrosis.