Original Contribution

Human Cytokine response to Texas crotaline envenomation before and after Antivenom administration

Patrick Crocker DO^a, Omid Zad MD^b, Truman Milling MD^b,?, Todd Maxson MD^a,

Benjamin King^b, Elbert Whorton PhD^c

^aDell Children’s Medical Center of Central Texas ^bUniversity Medical Center at Brackenridge ^cUniversity of Texas Medical Branch

Received 8 February 2009; revised 22 April 2009; accepted 23 April 2009

Abstract

Objective: The aim of this study was to characterize the human cytokine response to Texas crotaline envenomation before and after antivenom administration.

Methods: This study enrolled crotaline bite victims presenting to a regional trauma center and children’s hospital from March to November 2007 and age-matched unbitten controls. Blood spot cards were obtained from bite victims at presentation and at 1 and 6 hours after antivenom administration. One control sample was drawn from each of the age-matched controls selected from urgent care patients presenting for minor complaints. Samples were delivered to a laboratory using a proprietary method for quantitative evaluation of a large number of biomarkers in parallel with bead-based multiplex immunoassays.

Results: After obtaining informed consent, 14 crotaline bite victims (age range, 5-85 years; median age, 45 years; 50% female) (Snakebite Severity Score, 2-7; median, 3) and 14 age-matched controls were enrolled. There were 7 copperhead (Agkistrodon contortrix) bites, 4 rattlesnake (probably Western Diamondback Crotalus atrox) bites, 2 cottonmouth (Agkistrodon piscivorus) bites, and 1 bite from a snake that was not identified by the victim. In t tests, the means in the presentation samples for apolipoprotein A- I (Apo A-I), Apo C3, interleukin 4 (IL-4), myeloperoxidase, plasminogen activator inhibitor 1 (PAI-1), epidermal growth factor, and regulated upon activation, normal t-cell expressed and secreted were significantly lower and Apo H was significantly higher in the bite patients than in the controls. In the 1-hour sample, ?₁-antitrypsin, Apo A-I, Apo C3, eotaxin, IL-4, myeloperoxidase, and PAI-1 levels were lower and prostatic acid phosphatase and cancer antigen 125 levels were higher in the bite patients than in the controls. And in the 6-hour sample, ?₁-antitrypsin, Apo A-I, Apo C3, endothelin-1, IL-4, macrophage inflammatory protein 1?, myeloperoxidase, and epidermal growth factor levels were lower and Apo H level was higher in the bite patients than in controls (all P values b .05).

Conclusions: Crotaline venom produces a broad cytokine response in human bite victims. In particular, IL-4, myeloperoxidase, and Apo A-I and C3 levels remain altered despite antivenom therapy, whereas PAI-1 and regulated upon activation, normal t-cell expressed and secreted levels seem to normalize after antivenin as other markers are affected. Understanding this profile and further study of the markers identified might lead to improved therapies and better prognostic indicators.

(C) 2010

* Corresponding author.

E-mail address: [email protected] (T. Milling).

0735-6757/$ - see front matter (C) 2010 doi:10.1016/j.ajem.2009.04.038

Introduction

Background

Little has been published on the human cytokine response to crotaline envenomation. Only 1 study has examined the levels of interleukins and other cytokines in human victims of Costa Rican pit viper bites [1]. No study has examined the human cytokine response to North American crotaline venom, much less how that response is modulated by the administration of antivenom.

Importance

An improved understanding of the human cytokine response might lead to better therapies and prognostic indicators.

Goals of this investigation

We characterize the human cytokine response to crotaline envenomation by measuring the following at presentation and at 1 and 6 hours after antivenom therapy: ?₁-antitrypsin,

Table 1 Markers on preexisting assay provided by Rules Based Medicine

apolipoprotein A-I (Apo A-I), Apo C3, Apo H, eotaxin, endothelin-1, interleukin 4 (IL-4), macrophage inflammatory protein 1? (MIP-1?), myeloperoxidase, epidermal growth factor (EGF), plasminogen activator inhibitor 1 (PAI-1), prostatic acid phosphatase, regulated upon activation, normal t-cell expressed and secreted (RANTES), cancer antigen 125 , and 75 other markers on a preexisting assay (see Table 1). This “shotgun” approach was used to identify potential markers of envenomation for future study.

Materials and methods

Study design

We collected blood spots on filter paper from patients who suffered crotaline envenomation presenting to our regional trauma center and children’s hospital from March to November 2007. All patients presented within 6 hours of bite (median, 2.9 hours; 95% confidence interval [CI], 2.04- 3.56 hours). Our goal was to collect samples at presentation, which, in this population, was roughly 3 hours after the bite,

1. Adiponectin	30. GST	60. Lymphotactin
2. ?1-antitrypsin	31. G-CSF	61. MDC
3. ?-Fetoprotein	32. GM-CSF	62. MIP-1?
4. ?-2 Macroglobulin	33. Growth hormone	63. MIP-1?
5. Apo A-I	34. Haptoglobin	64. MMP-2
6. Apo C-III	35. IgA	65. MMP-3
7. Apo H	36. IgE	66. MMP-9
8. ?-2 Microglobulin	37. IgM	67. MCP-1
9. BDNF	38. Insulin	68. Myeloperoxidase
10. C-reactive protein	39. IGF-1	69. Myoglobin
11. Calcitonin	40. ICAM-1	70. PAI-1
12. Cancer antigen 19-9	41. Interferon-?	71. PAPP-A
13. CA-125	42. IL-1?	72. PSA, free
14. Carcinoembryonic antigen	43. IL-1?	73. Prostatic acid phosphatase
15. CD40	44. IL-1 ra	74. RANTES
16. CD40 ligand	45. IL-2	75. Serum amyloid P
17. Complement 3	46. IL-3	76. SGOT
18. CK-MB	47. IL-4	77. Sex hormone binding globulin
19. Endothelin-1	48. IL-5	78. stem cell factor
20. Eotaxin	49. IL-6	79. Thrombopoietin
21. EGF	50. IL-7	80. Thyroid binding globulin
22. ENA-78	51. IL-8	81. Thyroid stimulating hormone
23. Erythropoietin	52. IL-10	82. tissue factor
24. ENRAGE	53. IL-12 p40	83. TIMP-1
25. Factor VII	54. IL-12 p70	84. TNF-?
26. Fatty acid binding protein	55. IL-13	85. TNF-?
27. Ferritin	56. IL-15	86. TNF-RII
28. Fibrinogen	57. IL-16	87. VCAM-1
29. FGF-basic	58. Leptin	88. VEGF
CK-MB indicates Creatinine kinase-MB; G-CSF	59. Lipoprotein (a) 89. von Willebrand factor , granulocyte colony-stimulating factor; GM-CSF, granulocyte-monocyte colony-stimulating factor.

and at 1 and 6 hours after CroFab (Crotalidae polyvalent immune Fab [Ovine]; Savage Labs, Melville, NY) admin- istration. These blood spots were frozen and stored until the end of the study period, at which point a preexisting assay for 89 human biomarkers (Rules Based Medicine, Austin, TX) was run (see Table 1). The assay contained all previously tested markers for crotaline envenomation, with the excep- tion of xanthine oxidase, a marker examined in one animal study [2], in addition to many other markers.

Also, the initial panels of hematologic laboratory results obtained were assessed for each patient who was bitten, when available, and abnormal results were flagged. Labora- tory results considered pertinent to the study included platelets, international normalized ratio, prothrombin time, partial thromboplastin time, fibrinogen, and D-dimer.

We chose blood spots for ease of collection, storage, and transport from sites that transfer snakebite patients to our facility. We were aware that this would complicate the assay process as the reference ranges of the markers extracted from filter paper had not been well established, so we collected age-matched control group data over the same time period from patients with minor complaints presenting to the minor care area of the emergency department. Obviously, these controls did not receive scores for snakebite severity, snake type, or units of CroFab administered and were not used in analyses that compared these variables to each other.

Assay

Rules Based Medicine donated its proprietary Compre- hensive Multi-Analyte Profiles of blood proteins, a bead- based, multiplex immunoassay technology that provides an efficient approach for performing a rapid assessment of large numbers of protein antigens. This technology performs up to

100 multiplexed, microsphere-based assays in a single reaction vessel by combining optical classification schemes, biochemical assays, flow cytometry, and advanced digital signal processing hardware and software.

Setting

The setting for this study was a regional trauma center and a children’s hospital in central Texas that together care for about 40 snake envenomations a year, most of which are from crotaline. About half the yearly tally consists of patients transferred from outlying facilities, often with antivenom administration already begun.

Selection of participants

Patients, adult and pediatric, suffering from crotaline envenomation were enrolled after consent. The Brack- enridge Hospital Institutional Review Board approved the study protocol.

Snake species

Central Texas is home to a wide variety of crotalines, although the most common species that bite humans are the copperhead (Agkistrodon contortrix), the cottonmouth or water moccasin (Agkistrodon piscivorus), and a few species of rattlesnake, most commonly the Western Diamondback (Crotalus atrox).

Degree of envenomation was measured with the Snake- bite Severity Score (SSS), as it is the only validated system, although 2 older, better-known Classification systems exist [3-5]. As in the original validation study of the SSS, the score was determined retrospectively from chart review.

Statistical analysis

• • 1. Markers in control vs experimental group

All cases were labeled according to whether blood spot determinations were made for each biomarker at presentation and at 1 and 6 hours after antivenom administration. The resulting biomarker means for controls and bite victims were then compared at each time period and statistical significance levels were determined using unpaired t tests for differences. For every marker showing a significant difference in means between the control and treatment groups, a one-way analysis of variance was performed to examine the influence of the marker on SSS, the primary clinical measure. Because every subject did not have measurements made at every time period, only controls who had an age-matched study subject at a given time period were used. This is why, although the control group has only 1 set of values, the control means change as different subsets are compared with matched study subjects at different time periods.

• • 1. Markers in experimental group vs hematologic laboratory results in experimental group

The number of total abnormal laboratory results for each patient was compared with overall variation in each of the significant biomarkers found earlier using analysis of variance tests that included relative time of draw (ie, t = 0, 1, and 6) as a second independent variable.

Results

Experimental group

Fourteen patients were enrolled, 7 of whom were male. Median age was 41 years (range, 5-85 years). Seven patients reported being bitten by copperheads (A contortrix); 2, by a cottonmouth (A piscivorus); and 4, by rattlesnakes (C atrox most likely, but 9 other species inhabit the area). One patient did not see the snake that bit him. However, the bite wound from the unidentified snake showed ecchymosis and coagulopathy consistent with crotaline envenomation. Of the 14 patients, 9 were transferred from outside facilities, 2 of

these did not receive CroFab and 1 received CroFab during transport from the scene by helicopter.

Control group

Fourteen controls were enrolled, 5 of whom were male. Ages were matched according to those collected in the experimental group, so the mean and range were the same.

The chief complaints for control patients were primarily localized pain (n = 10), postoperative wound check (n = 1), chest pain without cardiac event (n = 1), pregnancy (n = 1), and anxiety/nervousness (n = 1).

Markers in control vs experimental group

Several markers in the assay tested positive for differences between the control and experimental groups, with the markers

Table 2 Markers with statistically significant differences between snake-bitten patients and controls

Time		Bite patients				Control patients		P	Direction
		Mean SD	CI			Mean SD	CI
?1-Antitrypsin	0	0.8	0.16	0.7 to 1	1		0.18 0.9 to 1.1	N.05	NS
	1	0.9	0.1	0.8 to 0.9	1.1		0.2 0.9 to 1.2	b.05	Down
	6	0.9	0.2	0.7 to 1	1.1		0.3 0.9 to 1.3	b.05	Down
Apo A-I	0	0.4	0.12	0.34 to 0.53	0.6		0.16 0.48 to 0.73	b.05	Down
	1	0.4	0.1	0.3 to 0.5	0.6		0.1 0.5 to 0.7	b.05	Down
	6	0.4	0.1	0.3 to 0.5	0.7		0.2 0.5 to 0.8	b.01	Down
Apo C3	0	41	11.9	31.8 to 50.1	72		31.2 48 to 96.1	b.05	Down
	1	35.7	10.1	27.9 to 43.5	76.8		28.3 55 to 98.6	b.001	Down
	6	44.7	24.7	24 to 65.3	80		36.2 50 to 110.3	b.05	Down
Apo H	0	85.1	25.1	65.8 to 104.4	54.6		14.4 43.6 to 65.6	b.01	Up
	1	74.4	29.3	51.9 to 96.9	65.7		19.2 50.9 to 80.4	N.05	NS
	6	85.4	18.7	69.8 to 101.1	63.8		14.1 52.1 to 75.6	b.05	Up
Eotaxin	0	168.7	92	98 to 239.5	227.4		134.7 124 to 331	N.05	NS
	1	165.6	55.9	122.7 to 208.6	260		81.1 197.6 to 322.4	b.05	Down
	6	172	78.6	106.3 to 237.7	269		169 127.8 to 410.3	N.05	NS
Endothelin-1	0	7.9	5.7	3.5 to 12.3	12.4		13 2.4 to 22.4	N.05	NS
	1	5.2	5.1	1.3 to 9.2	14.5		12.3 5.1 to 24	N.05	NS
	6	4.3	4.3	0.7 to 7.9	22.7		11.3 13.2 to 32.2	b.001	Down
IL-4	0	34.9	13.9	24.2 to 45.6	66.1		10.1 58.3 to 73.9	b.001	Down
	1	45.3	14.1	34.5 to 56.1	66.8		11.8 57.7 to 75.8	b.01	Down
6 45.4 12.8 34.7 to 56					66.6		9.8 58.4 to 74.8	b.01	Down
MIP-1?	0	254.5	225	163.6 to 345.4	481.8		526.3 201.4 to 762.3	N.05	NS
	1	345.8	346.6	79.3 to 612.2	346.4		286.7 126 to 566.8	N.05	NS
6 178.5 63.8 125.1 to 231.8					373.4		207.9 200 to 547.2	b.05	Down
Myeloperoxidase	0	9792.2	2501.2	7869.6 to 11 714.9	21 542.2		7443.2 15 820.9 to 27 263.6	b.001	Down
	1	11 315.6	2329.1	9525.3 to 13 105.8	22 733.3		7607.4 16 885.8 to 28 580.9	b.001	Down
	6	11 013.8	2386.3	9018.8 to 13 008.7	22 928.6		5999.1 17 380.3 to 28 476.8	b.001	Down
EGF	0	544.4	449.9	198.5 to 890.2	1528		1043.5 725.9 to 2330.1	b.05	Down
	1	995.9	860.1	334.8 to 1657	1324.9		420.5 1001.7 to 1648.1	N.05	NS
	6	685.8	180	535.3 to 836.2	1316.8		310.5 1057.1 to 1576.4	b.001	Down
PAI-1	0	106.2	58.5	61.3 to 151.2	246.4		126.1 149.5 to 343.4	b.01	Down
	1	142	51.1	102.7 to 181.3	214.3		88.5 146.3 to 282.3	.05	Down
	6	157.9	49.4	116.6 to 199.3	229.8		99.1 146.9 to 312.6	N.05	NS
Prostatic acid phosphatase	0	2.9	2.6	0.9 to 4.9	1.9		1.3 0.9 to 2.9	N.05	NS
	1	5.3	3.2	2.8 to 7.8	2.4		1.3 1.4 to 3.4	b.05	Up
	6	4	1.4	2.8 to 5.2	2.7		1.7 1.3 to 4.1	N.05	NS
RANTES	0	34.1	23.2	16.2 to 51.9	65.7		37.3 37 to 94.4	.05	Down
	1	49.9	15.5	37.9 to 61.8	59.9		25.8 40.1 to 79.7	N.05	NS
	6	43.5	17.2	29.1 to 57.8	64.3		25.1 43.4 to 85.3	N.05	NS
CA-125	0	6.9	5.1	2.9 to 10.8	4.2		5.2 0.2 to 8.2	N.05	NS
	1	8.8	3.1	6.4 to 11.3	2.9		4.7 -0.6 to 6.5	b.01	Up
	6	6.4	3	3.9 to 9.0	3.8		5.6 -0.8 to 8.4	N.05	NS

NS indicates not significant.

Units of measure for markers: ?₁-antitrypsin and Apo A-I, mg/mL; Apo C3 and Apo H, ug/mL; endothelin-1 and IL-4, pg/mL; prostatic acid phosphatase and RANTES, ng/mL; CA-125, U/mL; eotaxin, MIP-1?, and EGF, pg/mL; PAI-1, ng/mL; and myeloperoxidase, ng/mL.

of snakebite victims either above or below those of the controls. See Table 2 and Fig. 1 for significant results on the 14 markers.

Markers in experimental group vs hematologic laboratory results in the experimental group

Two patients did not have any pertinent laboratory values drawn. The number of abnormal laboratory results covaried significantly with 2 of the biomarkers in snake-bitten subjects found to be significantly different from those of controls: ?₁-antitrypsin (P b .05) and PAI-1 (P b .05).

Other analyses

None of the markers tested showed a significant covariance with SSS or with the number of vials of CroFab

administered. However, SSS correlated significantly with units of CroFab administration and with the type of snake involved (P b .05). See Tables 3-5.

No adverse events, such as allergic response to the CroFab administration, were seen in the treated population.

Limitations

In a study of this many markers and data points, it is always possible that some associations will be found by chance. This is a preliminary model to identify markers that warrant further study. Markers may also vary by species of snake, but obtaining even a small sample for each species would require many years or many centers. Eight patients received CroFab before arrival at the hospital site (7 at other sites, 1 on a helicopter) and, as a result, did not have a t = 0

Fig. 1 Units of measure for markers: ?₁-antitrypsin and Apo A-I, mg/mL; Apo C3 and Apo H, ug/mL; endothelin-1 and IL-4, pg/mL; prostatic acid phosphatase, RANTES (ng/mL), and CA-125, U/mL; eotaxin, MIP-1?, and EGF, pg/mL; PAI-1, ng/mL; and myeloperoxidase, ng/mL.

Criterion Points

Pulmonary system No symptoms/signs 0 Dyspnea, minimal Chest tightness, mild or vague discomfort, or respirations of 20-25 1 Moderate respiratory distress (tachypnea, 26-40 breaths/min; Accessory muscle use) 2 Cyanosis, air hunger, extreme tachypnea, or Respiratory insufficiency/failure 3 Cardiovascular system No symptoms/signs 0 Tachycardia (100-125 beats/min), palpitations, generalized weakness, benign dysrhythmia, or hypertension 1 Tachycardia (126-175 beats/min) or hypotension, with systolic blood pressure N100 mm Hg 2 Extreme tachycardia (N175 beats/min), hypotension with systolic blood pressure b100 mm Hg, malignant dysrhythmia, 3 or cardiac arrest Local wound No symptoms/signs 0 Pain, swelling, or ecchymosis within 5-7.5 cm of bite site 1 Pain, swelling, or ecchymosis involving less than half the extremity (7.5-50 cm from bite site) 2 Pain, swelling, or ecchymosis involving half to all of extremity (50-100 cm from bite site) 3 Pain, swelling, or ecchymosis extending beyond affected extremity (N100 cm from bite site) 4 gastrointestinal system No symptoms/signs 0 Pain, tenesmus, or nausea 1 Vomiting or diarrhea 2 Repeated vomiting, diarrhea, hematemesis, or hematochezia 3 Hematologic symptoms No symptoms/signs 0 coagulation parameters slightly abnormal: PT, b20 s; PTT, b50 s; platelets, 100 000-150 000/mL; or fibrinogen, 100-150 ug/mL 1 Coagulation parameters abnormal: PT, b20-50 s; PTT, b50-75 s; platelets, 50 000-100 000/mL; or fibrinogen, 50-100 ug/mL 2 Coagulation parameters abnormal: PT, b50-100 s; PTT, b75-100 s; platelets, 20 000-50 000/mL; or fibrinogen, b50 ug/mL 3 Coagulation parameters markedly abnormal, with serious bleeding or the threat of spontaneous bleeding: unmeasurable PT or PTT; 4 platelets, b20 000/mL; or undetectable fibrinogen; severe abnormalities of other laboratory values also fall into this category Central nervous system No symptoms/signs 0 Minimal apprehension, headache, weakness, dizziness, chills, or paresthesia 1 Moderate apprehension, headache, weakness, dizziness, chills, paresthesia, confusion, or fasciculation in area of bite site 2 Severe confusion, lethargy, seizures, coma, psychosis, or generated fasciculation 3

PT indicates prothrombin time; PTT, partial thromboplastin time. Points are assessed on the basis of manifestations caused by the venom itself (antivenom reactions not included). Ranges given are for adults; appropriate compensation should be made for age. Data from Dart et al [3].

draw performed. Although these transfers are usually rapid, we cannot be sure of the exact time of CroFab administration.

Table 3 The Snake bite Severity Score

Discussion

The study of the human host response to crotaline venom is in its infancy. The most commonly used antivenom, CroFab, has been shown to improve clinical outcomes [6,7], but its effect on the cytokine response has not been studied.

Known venom properties

Crotaline venom contains a complex mixture of enzymes, amino acids, lipids, and metals such as zinc, copper, and

magnesium [8,9]. The exact composition varies depending on several factors, such as species, age of snake, diet, season, and geographic location [8,10-13]. The enzymes possess mainly cytotoxic, hemorrhagic, and neurotoxic properties. The proteolytic enzymes, collagenase and hyaluronidase, produce the marked swelling and local tissue destruction at the site of envenomation [8]. Lysis of red blood cells, platelets, and mitochondria may be the direct result of phospholipase activation in the presence of lecithin and divalent metal ions [8]. Several different types of hemor- rhagins have also been identified. Direct lytic factor, phos- pholipases, and proteases may act synergistically with the hemorrhagins, resulting in extravasation of blood, ecchymo- sis at the bite site, and, possibly, systemic hemorrhage [8].

Crotaline venom exerts “thrombin-like” effects on fibrinogen molecules. Normally, thrombin forms fibrin by

Study ID	SSS	CroFab units	Length of stay (d)	Snake type
1	4	18	2	Unknown
2	4	0	1	Copperhead
3	4	12	3	Copperhead
4	5	12	3	Rattlesnake
5	2	4	b1	Copperhead
6	3	24	1	Cottonmouth
7	2	10	b1	Copperhead
8	3	6	4	Cottonmouth
9	2	5	1	Rattlesnake
10	7	14	3	Rattlesnake
11	3	6	1	Copperhead
12	7	9	2	Rattlesnake
13	2	4	1	Copperhead
14	2	4	1	Copperhead

removal of the low-molecular-weight peptides, fibrinopep- tide A and fibrinopeptide B, from each molecule of fibrinogen. Thrombin then stimulates fibrin stabilizing factor (factor XIII), adding strength to the fibrin meshwork forming a clot. Crotaline venom catalyzes the hydrolysis of an arginine sequence of the ? chain of fibrinogen, thereby splitting off fibrinopeptide A but not fibrinopeptide B [14,15]. The resulting monomers aggregate normally but form unstable, end-to-end linking instead of normal cross-linking fibrin polymers. These fibrin polymers are susceptible to normal fibrinolysis and phagocytosis by the reticuloendothelial system, and their presence is represented clinically by falling Fibrinogen levels and increased fibrin- fibrinogen degradation products. It has been shown that the rapid infusion of crotaline venom, as seen after a direct Intravenous envenomation, mimics true thrombin activity leading to widespread intravascular coagulation [16,17]. Theoretically, the D-dimer would remain normal and other Fibrin degradation products increase, distinguishing the disseminated intravascular coagulation-like syndrome of crotaline envenomation from true disseminated intravas- cular coagulation. D-dimer is unique compared with other fibrin degradation products in that it is the product of degradation of a true clot in which the fibrin has been properly cross-linked by factor XIII [18]. This dichotomy is not always born out in vivo; for example, 2 patients in our sample with SSS of 4 and 7 had increased D-dimers.

Table 4 SSS, CroFab, and snake type by patient

Previous envenomation biomarker research

interleukin 6, IL-1B, and matrix metalloproteinase-9 have been found to be increased in assays of muscle specimens from mice injected with Bothrops venom components [19], and serum samples from mice injected with Bothrops venom showed elevations of tumor necrosis factor ? (TNF-?), IL-1, IL-6, IL-10, and interferon gamma

[20]. However, the lone human study of 18 Costa Rican children bitten by Bothrops species found increases in IL-6, IL-8, TNF-?, MIP-1?, and RANTES [1]. Our results seem only to confirm the involvement of MIP-1? and RANTES in Texas crotaline envenomation, and we found them significantly reduced rather than increased.

Macrophage inflammatory protein 1? is a proinflamma- tory cytokine involved in activating granulocytes [21].

Regulated upon activation, normal t-cell expressed and secreted is also a Proinflammatory cytokine broadly studied in many conditions. It recently emerged in the trauma literature as a potential marker of traumatic brain injury severity [22].

Apolipoproteins A1 and C3

The potential involvement of Apo C3 in the venom response is also relatively unstudied, with the possible exception of a tangential in vitro study [23]. Apolipoproteins A-I and C3 have been studied extensively in coronary heart disease [24-26].

Apolipoprotein H

Also called ?₂-glycoprotein I, Apo H has been heavily studied as one of the phospholipids against which antibodies are formed in Antiphospholipid syndrome [27]. Antibodies to Apo H have lupus anticoagulant properties, meaning, in vitro, they prolong clotting times, but in vivo, patients with these antibodies are paradoxically prone to thrombosis [28]. Further study is required to determine the significance of the high levels of Apo H at presentation in our sample, which then fell at 1 hour and rose again at 6 hours.

Plasminogen activator inhibitor 1

The effects of venom on PAI-1 levels, which fell and then normalized after CroFab administration, are intriguing because they seem to mimic the effects of ancrod, a Malayan pit viper venom derivative in clinical trials for stroke [29], and activated protein C (Xigris), used in septic shock [30], both procoagulants known to cause PAI inhibition. Texas crotaline venom may have similar properties.

Epidermal growth factor, which plays an important role in the regulation of cell growth, proliferation, and

Table 5 SSS by snake type

Snake type	n	SSS range	SSS mean
Copperhead	7	2-4	2.71
Cottonmouth	2	3	3.00
Rattlesnake	4	2-7	5.25
Total	13	2-7	3.54

differentiation, has been shown to be modulated by crotoxin, a snake venom phospholipase A2 toxin [31].

Endothelin-1, a mediator of vascular permeability and constriction, was lower in the 6-hour sample. Snake venom, including some crotalines, contains sarafotoxins, which are structurally similar to endothelins and can activate the same receptor [32].

a₁-Antitrypsin

?₁-Antitrypsin, found significantly lowered in 1- and 6- hour samples, has been studied in vitro and shown resistant to some viper venom [33], although it seems that it may not be to local crotaline venom.

Other markers

Two markers, IL-4 and myeloperoxidase, remained significantly lower in all samples compared with controls. Interleukin 4 stimulates both B and T lymphocytes and augments their cytotoxic activity. A key regulator in humoral and adaptive immunity, it is a cytokine that induces differentiation of naive helper T cells (T_H0 cells) to T_H2 cells. Upon activation by IL-4, T_H2 cells subsequently produce additional IL-4. Interleukin 4 induces B-cell class switching to immunoglobulin E (IgE) and up-regulates major histocompatibility complex class II production [34-40]. Its importance in the cytokine response here is uncertain, as is that of myeloperoxidase, an enzyme involved in neutrophils’ respiratory burst, other than they may be signs of inflammation unaffected by CroFab. Eotaxin, an attractant for eosinophils, down in the 1-hour sample, is of uncertain significance, as are prostatic acid phosphatase, a marker for prostate cancer, and CA-125, a marker for ovarian cancer, both increased in the 1-hour sample.

Biomarker

• • 1. Hematologic laboratory results correlation

Only 2 biomarkers covaried with hematologic abnormal- ities, ?₁-antitrypsin and PAI-1. One should note that these laboratory result abnormalities are not the only drivers of antivenom administration, as advancing local signs would also indicate intervention. It seems reasonable that some markers, such as PAI-1 (particularly interesting given its role in coagulation), might predict hematologic abnormalities and others, such as the other markers that attained significance here, might predict local signs.

• • 1. Clinical course correlation

None of the markers showed the desired correlation with snakebite severity as measured by the SSS. This may have been due to the way the SSS was calculated, that is, as a global assessment of severity during hospital course. In future studies, we plan to calculate an independent SSS at the time each sample is drawn. Markers also showed no

direct correlation to the number of CroFab units administered, an indirect measure of severity of illness course, a preliminary disappointment in the search for a marker exhibiting a dose-dependent rise with envenoma- tion, but ? error is imminently possible here given the small sample size.

Conclusion

Crotaline venom produces a broad cytokine response in the human host, which seems to be modulated by antivenom administration. Use of the Multi-Analyte Profile allowed us to study many markers in parallel, not only confirming prior reports of the involvement of RANTES and MIP-1? but also identifying several other markers, such as apolipoproteins, endothelin-1, myeloperoxidase, IL-4, PAI-1, ?₁-antitrypsin, and EGF, which may warrant further study.

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