Interleukin-8 Expression in AIDS-Associated Lymphoma B-Cell Lines

Biochemical and Biophysical Research Communications 282, 369 –375 (2001) doi:10.1006/bbrc.2001.4579, available online at http://www.idealibrary.com on

Interleukin-8 Expression in AIDS-Associated Lymphoma B-Cell Lines Venkatanarayanan Sharma 1 and Li Zhang Laboratory of Cytokine Research, Department of Biology, University of West Florida, Pensacola, Florida 32514

Received February 12, 2001

Interleukin 8 (IL-8), a member of the CXC subfamily of chemokines, is a potent inflammatory cytokine produced by many cell types in response to several stimuli. In an attempt to determine whether human B-cell IL-8 functions as an autocrine growth factor, a wide panel of B-cell lines derived from patients with AIDSassociated B-cell lymphomas (AABCL) (n ⴝ 5) and from non-AABCLs (n ⴝ 8) was studied for expression of IL-8, IL-8 Receptor type A (IL-8R), and secretion of IL-8 protein. Using RT-PCR and Northern Blot analysis, we were able to observe IL-8 expression ubiquitously. However, IL-8R expression was seen only in EBV negative (4 out of 7) B-cell lines. EBV and HIV-1 activated B-cell line; HBL-1, was the major secretor of IL-8. Our results demonstrate that IL-8 is expressed in malignant B-cell phenotypes that correspond to a narrow window in the B-cell differentiation pathway (pre-B, early-B, and intermediate-B) as well as in normal CD19-enriched B-cells. Furthermore, IL-8 autocrine loops were not evident since IL-8R was detected only in cell lines that did not secrete IL-8 protein. © 2001 Academic Press

Key Words: interleukin-8; B-cell lines; AIDS; lymphoma; chemokine; RT-PCR.

Interleukin 8 (IL-8) is a member of the superfamily of inducible, secreted, pro-inflammatory cytokines commonly referred to as chemokines. These cytokines are characterized by four-conserved cysteine residues and have been shown to chemoattract and activate different leukocyte subsets (1). These chemokines, based on the positioning of the cysteine residues are further divided into two subfamilies. The “␣” or C-X-C subfamily, which has the first two cysteine residues separated by a variable amino acid that includes IL-8, GRO/MGSA, platelet factor 4, ␤-thromboglobulin and IP-10. The “␤” or C-C subfamily, has the first two cysteine residues adjacent and consists of members like RANTES, MIP-1␣, MIP-1␤, 1

To whom correspondence should be addressed. Fax: (850)-4742749. E-mail: [email protected].

MCP-1 and I-309. The genes for the C-X-C and C-C chemokines are located on chromosome 4q and 17q, respectively. The C-X-C chemokines preferentially chemoattract and activate neutrophils, whereas the C-C chemokines are chemotactic for monocytes and T cells (1– 4). IL-8 exerts its biological function by binding to specific cell surface receptors. Two distinct but homologous human IL-8 receptors designated type A (IL-8R) or type B have been cloned and IL-8 can bind both receptors with high affinity (5, 6). IL-8 also binds to a multispecific chemokine receptor identified on the surface of the red blood cells (7). Many cell types including monocytes/macrophages, T cells, neutrophils, fibroblasts, endothelial cells, keratinocytes, hepatocytes, astrocytes, and chondrocytes can produce IL-8 in response to a wide variety of proinflammatory stimuli such as exposure to IL-1, TNF, LPS, and viruses (1). The potent pro-inflammatory effect of IL-8 is not restricted only in chemoattracting neutrophils. IL-8 has also been shown to be chemotactic for basophils, T cells and eosinophils (1). IL-8 is reported to enhance the adherence of neutrophils to endothelial cells and subendothelial matrix proteins by inducing the expression of CD11/CD18 (8). In keratinocytes IL-8 is shown to function as a co-mitogen (9). Schadendorf et al. had reported autocrine activity for IL-8 in melanoma cells (10). IL-8 is also reported to stimulate angiogenesis in vivo (11). Elevated IL-8 levels have been reported in rheumatoid arthritis (12) and in a variety of inflammatory diseases. In this report we describe the constitutive and mitogen induced expression and secretion of IL-8, and expression of IL-8R in human B-cells derived from patients with Burkitt’s lymphoma and AIDS-related Burkitt’s lymphoma. Since IL-8R was not detected in any of the cell lines secreting IL-8 protein constitutively, our results suggest no apparent evidence for IL-8 autocrine loops. MATERIALS AND METHODS Cell lines. The study included thirteen tumor B-cell lines derived from patients with undifferentiated lymphomas of Burkitt’s and

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non-Burkitt’s origin, as described previously (13, 14). The characteristics of these cells are summarized in Table 1. Five of these cell lines—10C9, 2F7, HBL-1, HBL-2, and HBL-3—are AIDS associated B-cell lines (AABCL). 10C9 is an EBV ⫹ human B-cell line established from a non-Hodgkin’s lymphoma patient with AIDS. 2F7 is also EBV ⫹, and was established from a Burkitt’s lymphoma patient with AIDS. HBL-1, HBL-2, and HBL-3 were derived from patients with AIDS-associated lymphomas and were a kind gift from Dr. Riccardo Dalla-Favera (Columbia University, NY). HBL-1 is EBV ⫹, whereas HBL-2 and HBL-3 are EBV ⫺ (15). HIV-1 viral sequences were not detected in any of these cell lines. The non-AABCLs comprised of five EBV ⫺ American Burkitt’s lymphoma cells lines: BJAB, EW36, CA46, ST486 and MC116, and three EBV ⫹ African Burkitt’s lymphoma cell lines Raji, Namalva, and Daudi. Establishment of the B-cell lines together with their phenotypic characteristics has been reported previously (13, 14, 16, 17). All cell lines were obtained from American type culture collection (ATCC, Rockville, MD) and were maintained in suspension culture in RPMI 1640 with 10% fetal calf serum (FCS) (Cellgro; Fisher Scientific, Pittsburgh, PA) at 37°C in 5% CO 2 humidified atmosphere. Exposure of cells to phorbol 12-myristate 13-acetate. Cells obtained on day four of subculture were resuspended in fresh medium at a cell density of 1 ⫻ 10 6 cells/ml. The cells were incubated in 75-cm 2 tissue culture flasks at 37°C for various lengths of time in the presence or absence of the tumor promoter phorbol 12-myristate 13-acetate (PMA; Sigma Chemical Co., St. Louis, MO). A wide range of PMA concentrations (5–100 ng/ml) were studied, and optimal results were obtained at a concentration of 10 ng/ml, as described previously (13, 14, 16, 17). Control and PMA-activated cultures were harvested at designated time points, and the cells were immediately processed for RNA isolation. The supernatants were collected and stored at ⫺80°C until assayed for IL-8 secretion. Northern blot analysis. Total cellular RNA was extracted from control and PMA-activated cells using a guanidinium isothiocyanate/ phenol chloroform methodology, and poly(A) ⫹ RNA was prepared using the Invitrogen Fast Track mRNA isolation kit (Invitrogen, San Diego, CA) following the manufactures direction, and as described previously (13, 14, 16, 17). Total RNA samples (10 ␮g) were analyzed by electrophoresis through 1% agarose-formaldehyde gels followed by northern blot transfer to GeneScreen Plus membranes (New England Nuclear, Bannocburn, IL). The gels were stained with ethidium bromide to confirm that approximately equivalent amounts of RNA were loaded in each lane. RNA blots were prehybridized at 42°C for 2 h in a solution containing 50% formamide, 5⫻ SSC (1⫻ SSC ⫽ 150 mM NaCl ⫹ 15 mM sodium citrate), 1% SDS, 1⫻ Denhardt’s solution (0.02% Ficoll, 0.02% polyvinylpyrrolidone, 0.02% BSA), and 1 mg of sheared salmon sperm DNA/ml. Hybridization for at least 18 h at 42°C was done in a similar solution in which the DNA and SDS were replaced with 0.02 mg of Escherichia coli tRNA/ml and ⬎10 6 cpm of random prime-labeled probe. The IL-8 cDNA probe (18) (a kind gift from Dr. J. J. Oppenheim, NCI, Frederick, MD) was labeled by the Random Primer labeling Kit (Boehringer-Mannheim, Indianapolis, IN) with [␣- 32P]dCTP (3000Ci/mmol, Amersham Corp., Arlington Heights, IL). Membranes were washed twice in 2⫻ SSC and 0.1% SDS at 25°C for 15 min, twice in 2⫻ SSC and 0.1% SDS at 60°C for 30 min. Membranes were placed at ⫺70°C and exposed to Hyperfilm (Amersham), as described previously (16, 17). Reverse transcription-polymerase chain reaction (RT-PCR) analysis for detection of interleukin-8 and interleukin-8 receptor transcript. Using a Perkin–Elmer PE2400 Thermal Cycler (Perkin–Elmer, Foster City, CA), cDNAs were prepared by reverse transcription (RT) of 0.1 ␮g of poly(A)⫹ RNA or 1 ␮g of total RNA, as described previously (13, 14, 16, 17 19 –22). Briefly, 5 ␮l of the cDNA reaction were amplified with 2.5 U of Taq DNA Polymerase (Fisher). The reaction mixture consisted of 20 pmol each of the forward and reverse primers in PCR buffer, 2 mM MgCl2, and 200 ␮M each of dATP, dCTP, dGTP, and dTTP. The mixture was amplified over 35 cycles. The first two cycles for IL-8 amplification

consisted of denaturing at 97°C for 1 min, primer annealing at 58°C for 1 min, and primer extension at 72°C for 1 min. An additional 33 cycles of denaturation at 94°C for 1 min were followed with similar annealing and primer extension conditions as used in the first two cycles. However, for IL-8R the annealing temperature was 60°C, followed by similar PCR conditions as mentioned above. A final extension was performed at 72°C for 7 min, followed by storage at 4°C. Specific primers for the upper and lower strands for IL-8 consisted of 5⬘CGATGTCAGTGCATAAAGACA-3⬘ and 5⬘-TGAATTCT CAGCCCTCTTCAAAAA-3⬘, respectively, with an expected amplicon size of 200-bp, as published previously (20). Primers for the upper and lower strands for IL-8R consisted of 5⬘-GGCCGACCTG CTTTTTGC-3⬘ and 5⬘CCGCAGCACCATGCGCATTT-3⬘, respectively, with an expected amplicon size of 361-bp (5). The house keeping gene glyceraldehyde-3phosphate dehydrogenase (GAPDH) was amplified concurrently with IL-8 and IL-8R using the same RT mixture. The primers used for GAPDH amplification have been published previously (13, 14, 16, 17 19 –22). Primers were custom made by Bioserve (Laurel, MD). The amplicons were detected by ethidium bromide staining after electrophoresis through 2% Tris-borate/EDTA agarose gels. Semiquantification of relative intensities of the PCR amplicons was performed using the Collage 3.0 intensity scanning function (Macintosh, Apple Computer Inc., Cupertino, CA). Intensity readings were quantified by comparing the IL-8/IL-8R expression levels with those of GAPDH. Cloning and sequencing of RT-PCR products. The 200-bp amplicon of IL-8 and the 361-bp amplicon of IL-8R from three representative cell lines, HBL-1, HBL-2 and Raji were subcloned into the dephosphorylated HincII site of the pBluescript II plasmid vector (Stratagene, La Jolla, CA) following modifications of T4 DNA polymerase to remove the 3⬘ non-template-directed base addition, as described previously (13, 14, 16, 17 19 –22). Briefly, the blunt-end amplicons were treated with T4 polynucleotide kinase and ligated into the vector using T4 DNA ligase at 12°C overnight. After transformation of competent bacteria, white colonies growing in LuriaBertani medium (LB) agar containing 100 ␮g ampicillin/ml, 40 ␮g 5-bromo-4-chloro-3-indoyl-␤-D-galactoside/ml were selected and grown overnight in 5 ml of terrific broth containing 100 ␮g ampicillin/ml. Plasmid mini preparations were performed by alkali lysis of 1.5 ml of the overnight culture. Plasmids containing the appropriate size inserts, as determined by electrophoresis through 1.5% Tris acetate/EDTA agarose gels after restriction digestion were sequenced using the Applied Biosystems Sequencer model 373A (Applied Biosystems, Foster City, CA). IL-8 enzyme-linked immunosorbent assay (ELISA). Supernatants obtained from control and PMA activated cells were collected and 100 ␮l of supernatant was assayed. After normalizing for equal amounts of protein, secreted IL-8 was detected using the Quantikine IL-8 immunoassay (R&D Systems, Minneapolis, MN), following the manufacturer’s guidelines. The minimum detectable dose without apparent cross-reactivity to various cytokines was ⬍10 pg/ml, as outlined by the manufacturer.

RESULTS Detection of IL-8 Transcript by Using RT-PCR and Northern Blot HBL-1 cell line was used as a representative B-cell line to study the kinetics of IL-8 mRNA transcripts and secretion from 1 to 96 h. HBL-1 cells obtained on day three after subculture were resuspended at 5 ⫻ 10 5 cells/ml in fresh medium with 10% FCS and incubated at 37° with and without PMA for 1, 3, 5, 8, 24, 48, 72, and 96 h. At each time point, the culture was harvested for RNA isolation, and the supernatants were collected

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Characteristics of B-Cell Lines Cell line

AABCL American Burkitt’s lymphoma Non-AABCL

African Burkitt’s lymphoma Non-AABCL

EBV status

Continent

Histology

Tissue source

10C9 2F7 HBL-1 HBL-2 HBL-3 BJAB EW36 CA46 ST486 MC116

Positive Positive Positive Negative Negative Negative Negative Negative Negative Negative

N. America N. America N. America N. America N. America N. America N. America S. America N. America N. America

Burkitt’s Burkitt’s UL* UL UL Burkitt-like UL Burkitt’s UL UL

Peripheral blood Peripheral blood Peripheral blood Pleural effusion Liver Lymphoblastoid Pleural effusion Ascitic fluid Ascitic fluid Pleural effusion

Raji Daudi Namalva

Positive Positive Positive

Africa Africa Africa

Burkitt’s Burkitt’s Burkitt’s

Jaw tumor Orbital tumor Lymphoblastoid

* UL, undifferentiated lymphoma.

for ELISA. Constitutive IL-8 transcripts were expressed from 1 h with a peak accumulation at 24 h. Furthermore, mitogen stimulation augmented mRNA

expression by approximately one fold compared to untreated control cells at each time point (data not shown). To ensure that the differences seen in mRNA

FIG. 1. Interleukin-8 (IL-8) mRNA expression in human B-cell lines as determined by using reverse transcription polymerase reaction (RT-PCR) analysis. (Left) Acquired immune deficiency syndrome (AIDS)-associated B-cell lines (AABCLs); (middle) non-AABCLs Epstein– Barr virus (EBV ⫺); and (right) non-AABCLs, EBV ⫹. The 200-bp amplicon of IL-8 was generated from poly(A) ⫹ and total RNAs isolated from control and phorbol 12-myristate 13-acetate (PMA)-stimulated cells. The 358-bp amplicon represents GAPDH for semiquantitation by densitometry. 371

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expression at these time points were not caused by variations in PCR efficiency or relative amounts of amplicon products, but indeed reflected changes in IL-8 kinetics, these expressions were normalized to that of GAPDH and repeated several times. Although the constitutive secretion of IL-8 was detected as early as 1 h, increased secretion in control and PMA stimulated cells was observed at 24 h time point onwards. Having identified the optimum time point for IL-8 expression and secretion in the representative HBL-1 cell line as the 24 h time point, we performed RT-PCR analysis on constitutive and PMA-induced expression of IL-8 in all the other B-cell lines, several times. Constitutively all the thirteen cell lines (Table 1) expressed IL-8 mRNA transcript as shown in Fig. 1. However, upon mitogen stimulation with PMA, the IL-8 mRNA transcript levels in AABCLs reveal a 1.5-fold increase in HBL-1, a 2-fold increase in HBL-3 and no significant change in 1OC9, 2F7 and HBL-2 cell lines when normalized to the amplicons of the housekeeping gene, GAPDH. Among EBV-negative non-AABCLs, PMA induced IL-8 expression by a 0.6-fold for EW36, a 0.5-fold for CA46, and a 0.8-fold for ST486. However, no apparent changes were observed in BJAB and MC116. In EBV-positive African Burkitt’s lymphoma B-cell lines, PMA induced increased IL-8 expression in Raji cells by a 1.5-fold and that of Namalva cells by a 3-fold. However, with Daudi cells we did not observe any change in IL-8 transcript as shown in Fig. 1. In essence, ubiquitous constitutive expression of IL-8 mRNA was observed in every tumor derived B-cell line attempted in this study. EBV presence or absence does not appear to be a factor for IL-8 expression, since both EBV ⫹ and EBV ⫺ cell lines expressed IL-8 mRNA. Two representative cell lines Raji and HBL-1 were cultured in the absence (control) or in the presence of 10 ng/ml of PMA. Total and poly(A) ⫹ RNA was isolated from control and PMA stimulated cells after 24 h of culture for Northern blot analysis. A single band of 1.8 kb that corresponds to the IL-8 mRNA was constitutively expressed by both the cell lines. PMA stimulation augmented IL-8 expression as shown in Fig. 2. The housekeeping gene, GAPDH (1.4 kb) was analyzed as an internal control. These results further support and confirm our RT-PCR data on IL-8 fold expression. Detection of IL-8R Transcript by RT-PCR To investigate possible autocrine loops for IL-8 in these cell lines we looked for IL-8R expression. HBL-2 cell line was used as a representative B-cell line to study the kinetics of IL-8R mRNA transcripts from 1 to 96 h. As observed with IL-8, IL-8R transcripts were also expressed from 1 h with a peak accumulation at 24 h (data not shown). Figure 3 displays IL-8R expression in all the 13-cell lines studied at the 24 h time point. As shown in Fig. 3, IL-8R transcripts were ob-

FIG. 2. Analysis of interleukin-8 (IL-8) mRNA expression as determined by using Northern blot analysis. Two representative B-cell lines Raji and HBL-1 were cultured in the absence (control) or the presence of 10 ng/ml of phorbol 12-myristate 13-acetate (PMA). Total RNA was isolated from control and PMA-stimulated cells after 24 h of culture. IL-8 mRNA (1.8 kb) was constitutively expressed by both the cell lines that was further augmented by PMA. The housekeeping gene GAPDH (1.4 kb) was analyzed as an internal control.

served only in HBL-2 of the AABCLs. However, in the non-AABCLs EBV-negative cell lines, IL-8R transcripts were detected in three (EW36, ST486 and MC116) out of five cell lines, both constitutively and upon PMA induction. In the non-AABCL EBV-positive cell lines, we could not detect IL-8R transcript either constitutively or upon PMA induction. These results demonstrate that IL-8R is seen only in EBV-negative B-cell lines of both AABCL and non-AABCLs. Cloning and Sequencing IL-8, IL-8R, and GAPDH RT-PCR Products We cloned and sequenced the 200-bp amplicons of IL-8, 361-bp amplicons of IL-8R, and the 358-bp of GAPDH from three representative cell lines, HBL-1, HBL-2 and Raji. The IL-8 and IL-8R sequences were identical to the published sequences (2, 5). The GAPDH sequence was identical to sequence previously described (13, 14, 16, 17, 19 –22; data not shown). IL-8 ELISA Secretion of IL-8 was quantified using standard IL-8 protein provided by the manufacturer (R&D Systems). Least-square fit linear regression analysis was performed on the absorbance value measured by using a

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FIG. 3. Interleukin-8 receptor type A (IL-8R) nRNA expression in human B-cell lines as determined by using reverse transcription polymerase reaction (RT-PCR) analysis. (Left) Acquired immune deficiency syndrome (AIDS)-associated B-cell lines (AABCLs); (middle) non-AABCLs Epstein–Barr virus (EBV ⫺); and (c) non-AABCLs, EBV ⫹. The 361-bp amplicon of IL-8R was generated from poly(A) ⫹ and total RNAs isolated from control and phorbol 12-myristate 13-acetate (PMA)-stimulated cells. The 358-bp amplicon represents GAPDH for semiquantitation by densitometry.

DYNEX-MRII microplate autoreader (Dynatech Laboratories, Inc., Chantilly, VA), and the results are summarized in Table 2. Significant IL-8 protein secretion were observed in 2F7 and HBL-1 cells (AABCL), EW36

and CA46 cells (EBV-negative non-AABCL), and Raji and Namalva cells (EBV-positive non-AABCL) either constitutively or upon PMA stimulation, as shown in Table 2. However, we did not observe any specific pat-

TABLE 2

Analysis of Interleukin-8 (IL-8) Secretion by Human B-Cell Lines IL-8 secretion (pg/ml) Cell line

AABCL American Burkitt’s lymphoma cell lines Non-AABCL

African Burkitt’s lymphoma cell lines Non-AABCL

373

EBV status

Control

PMA

10C-9 2F7 HBL-1 HBL-2 HBL-3 BJAB EW36 CA46 ST486 MC116

Positive Positive Positive Negative Negative Negative Negative Negative Negative Negative

0 12.21 728.58 0 3.18 0 0 28.69 0 0

0 141.83 2757.83 0 7.38 0 235.12 77.37 3.18 0

Raji Daudi Namalva

Positive Positive Positive

200.29 0 116.38

467.89 1.28 306.93

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terns of IL-8 secretion between AABCLs, EBV ⫺ nonAABCLs, and EBV ⫹ non-AABCLs at the 24 h time point. The data as shown in Table 2, suggests heterogeneity in IL-8 secretion. DISCUSSION Ability of B-cells to recognize and respond to antigens requires close cell-to-cell communication involving factors like cytokines. AIDS-related B-cell lymphomagenesis is a complex process with aberrant cytokine/cytokine receptor modulations. We had previously identified several important cytokines like IL-7 (16), IL-10 (23), IL-12 (17), MIP-1␣ (13), and IL-16 (14) in B-cell lines. Some of these B-cell cytokines are demonstrated to function as autocrine growth factors (13). In this study we report that tumor derived human B-cell lines can express and secrete IL-8. Furthermore, a majority of EBV-negative B-cell lines express IL-8R mRNA. We also observed CD19 enriched B-lymphocytes to express and secrete IL-8 as reported by Nielsen et al. (24). However, we could not detect IL-8R transcripts suggesting no autocrine role for IL-8 in normal B-cells (data not shown). The roles of chemokines in the pathology of retroviral-associated diseases are not clearly known. Several reports, including our own have shown that virally encoded transcriptional activators, tax of HTLV and tat of HIV, are capable of inducing a number of chemokine genes. Based on the biological activities attributed to chemokines, including a role in inflammation, leukocyte recirculation, and other mechanisms of host defense, an understanding of chemokine mechanisms of action will be beneficial for treatment of various inflammatory and non-inflammatory disease states. In a previous report, we demonstrated that the induction of IL-8 by HTLV tax occurs by an autocrine mechanism (21). Thus, it is tempting to speculate that in HTLV associated rheumatoid arthritis (RA), MIP-1␣ (22) and IL-8 (21) may be important chemokines induced by HTLV tax. Similar to HTLV tax, HIV tat induced IL-8 expression (20) and secretion in both T and B-cell lines (data not shown). These findings suggest the possibility that modulations of viral expression may be influenced by some of these regulatory cytokines and vice versa. In addition, some of the soluble factors secreted by B-cells may in fact have suppressive effects on viral replication (13, 14). Although B cells are not natural targets for HIV, their transformation with EBV may cause expression of the CD4 molecule facilitating viral infection (25). In summary, we described here the expression and secretion of IL-8 in 13 tumor-derived B-cell lines. Although most of the EBV-negative B-cell lines expressed IL-8R, no apparent autocrine loops were evident for IL-8 in these B-cells, as they did not secrete significant amounts of IL-8 protein, constitutively. In our experience the IL-8 secretor cell lines did not express IL-8R

and the cell lines that expressed IL-8R did not secrete IL-8 protein. In an attempt to induce IL-8 autocrine loop, we used PMA to trigger IL-8 secretion in cell lines that express IL-8R. Only one cell line EW36, upon PMA induction secreted a significant amount of IL-8 protein, indicating a possibility of an autocrine loop upon mitogen stimulation. In conjunction with our previous reports that EBV and HIV-1 activated B cells are the major secretor of lymphokines, and given the pivotal role of IL-8 chemokine, it can be emphasized that these cytokines play an important role in the development of AIDS-associated B-cell lymphomagenesis. ACKNOWLEDGMENTS This work was partially supported by Small Grant Awards (Nos. 5302-293-22 and 5302-282-22) from University of West Florida, Pensacola, FL 32514 (V.S.). We thank John VanSteenbergen for assistance, John Blackie for the photographs, and Dr S. Bagui for statistical analysis.

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