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The Journal of Immunology, 1998, 161: 1306-1312.
Copyright © 1998 by The American Association of Immunologists

Human and Nonhuman Primate Lymphocytes Engrafted into SCID Mice Reside in Unique Mesenteric Lymphoid Structures1

Hollie Hale Donze2,*, James E. Cummins, Jr.*, Rebecca S. Schwiebert*,{dagger}, Patricia N. Fultz*, Susan Jackson* and Jiri Mestecky3,*,{ddagger}

Departments of * Microbiology, {dagger} Comparative Medicine, and {ddagger} Medicine, University of Alabama, Birmingham, AL 35294


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The present study compares the location and phenotype of B lineage lymphocytes in tissues from SCID mice engrafted with PBMC of human, chimpanzee, and pig-tailed macaque origin. In mice repopulated with both human and nonhuman primate lymphocytes, plasma cells were found in the peritoneal cavity in vascularized structures located in the mesentery near the pancreas, intestines, and spleen. The predominant isotype of the plasma cells was IgG; IgM and IgA cells were also present. {kappa} and {lambda} light chains were expressed by 62% and 38% of the Ig-containing cells, respectively. J chain expression occurred in most cells irrespective of the Ig isotype. In the SCID mice engrafted with human lymphocytes, a few IgM-containing cells were found in the spleen; plasma cells were not found in other tissues, including the intestine. The aggregation of plasma cells did not appear to be a result of infection with EBV. T cells were rarely found in the lymphoid aggregates but were recovered from the spleen and peritoneal lavage. Human Ig levels in the serum of engrafted mice reflected the isotype distribution of the cells with IgG > IgM >= IgA.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Severe combined immune deficient (SCID) mice, described in 1983 (1), are homozygous for a mutation in the XRCC7 gene, which encodes a DNA-protein kinase catalytic subunit (DNA-PKcs). The lack of protein kinase activity renders these mice without functional B and T cells due to impaired V(D)J recombination (2, 3). These immunodeficient mice can be reconstituted with lymphocytes from other strains of mice as well as from other species, including humans (1, 4, 5). Two models for reconstitution of SCID mice with human cells have been developed: the SCID-hu model (4), which utilizes stem cells found in fetal tissue engraftments to reconstitute the SCID immune system, and the hu-PBL-SCID model (5), in which PBMC are injected into the peritoneal cavity or the tail vein.

Engrafted SCID mice have been used to study responses of the human immune system to infections with HIV and EBV, bacterial infections, and vaccines, as well as to determine lymphocyte homing patterns (6-8). Previous work using the hu-PBL-SCID model has shown that the majority of human cells remain in the peritoneal cavity, repopulating only the spleen, thymus, and lymph nodes (5, 9, 10). Human Igs are present in the serum and peritoneal lavage fluids of the mice, indicating engraftment of, and differentiation into, Ig-producing plasma cells (7, 11, 12, 13, 14, 15, 16). Long-term B cell engraftment may be influenced by the EBV status of the donor. In mice repopulated with lymphocytes from EBV+ donors, B cell lymphomas appeared after 8 wk (11, 17, 18, 19). However, early after reconstitution of SCID mice with human B cells, the phenotype and activity of the B cells can be compared with those of the normal human immune system (8, 12, 14, 20).

While several groups have studied B cell populations in the hu-PBL-SCID model, mice that have been engrafted with nonhuman primate B cells have not been characterized. Because nonhuman primates are often infected with pathogens similar to those of humans, SCID mice repopulated with nonhuman primate lymphocytes would be an effective tool for direct comparisons between human and nonhuman primate immune responses to vaccines developed against these pathogens. To determine the early engraftment properties of B cells in SCID mice, we investigated the location, phenotype, and EBV status of human B cells 3 weeks after repopulation with human PBMC. In addition, we obtained tissues from nonhuman primate PBMC-engrafted SCID mice for comparisons of primate and human B cell repopulation in SCID mice.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals

C.B-17 (scid/scid) mice were obtained from the University of Alabama at Birmingham Animal Resources Program. The mice were maintained in cages fitted with microisolators. Cages, bedding, food, and water were autoclaved before use, and no antibiotics were administered.

Engraftment of SCID mice with PBMC

Venous blood was collected from seven healthy adult human donors. PBMC were isolated by centrifugation on a Ficoll-Hypaque density gradient (Sigma, St. Louis, Mo). The cells were washed three times in Dulbecco’s PBS and resuspended in RPMI 1640 containing 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Mediatech, Herde, VA). PBMC were treated for 2 days in culture with either the addition of 0.5% PWM (Life Technologies, Gaithersburg, MD), 10 ng/ml PMA (Sigma), or 0.1% PHA (Difco, Detroit, MI). The cells were then washed extensively, and viability was determined by trypan blue dye exclusion. The cells were resuspended at 2 to 3 x 107 cells/250 to 1000 µl in RPMI 1640.

To enhance engraftment, six mice were pretreated 18 h before receiving unstimulated or stimulated human PBMC by i.p. administration of 20 µl of anti-asialo GM1 ganglioside (Wako Bioproducts, Richmond, VA) to remove NK cells (21, 22). Human growth hormone (5 µg HuGH,4 Protropin, Genentech, San Francisco, CA) was administered to each animal 18 h after injection of donor cells, and the HuGH treatment continued every other day until the animals were sacrificed. A total of 22 mice were injected i.p. with 2 to 3 x 107 freshly isolated or cultured human PBMC (Table IGo). Mice were sacrificed 3 to 4 wk later. One mouse died during the study and only two hu-PBL-SCID mice were found to have cells containing murine Igs (Table IGo).


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  		Table I. Protocols for hu-PBL-SCID mice

 
Sample collection

One day before sacrifice, saliva was collected by pipette after stimulation by i.p. injection with 2 µg of carbamylcholine chloride (Carbocol, Sigma). After the animals were anesthetized, blood was collected by brachial bleed. The peritoneal cells were recovered by flushing the cavity with 3 ml of PBS. Bile was collected in disposable syringes into 100 µl of PBS. In all mice spleen, mesentery, and small intestine were harvested; in addition, liver, kidney, large intestine, thymus, pleural fat, lungs, salivary glands, peritoneal lining, and diaphragm from some animals were collected for histology.

Mesenteric tissues were obtained from SCID mice reconstituted with PBMC from chimpanzees (19 mice) and pig-tailed macaques (15 mice). PBMC from pig-tailed macaques were Con A stimulated and infected with SIVsmmPBj14 in vitro before engraftment, and each mouse received 2.5 x 107 to 5 x 107 cells i.p. (23). Mesenteric tissues from control age-matched BALB/c and C57BL/6 mice were also obtained at time of sacrifice.

Ig levels

Serum IgA, IgG, and IgM isotypes were determined by ELISA. Bile and saliva were analyzed for IgA. Briefly, 96-well polyvinyl microtiter plates (Dynatech, Chantilly, VA) were coated with the F(ab')2 fragments of goat Abs specific for human IgG (2.5 µg/ml), IgA (2.5 µg/ml), or IgM (1.0 µg/ml) (Jackson ImmunoResearch, West Grove, PA) to capture the respective Ig. Biotinylated goat anti-human IgA, IgG, and IgM (Tago Immunologics, Burlingame, CA) were used to detect bound Ig. A serum pool (Moni-Trol E, Baxter, McGaw Park, IL) with known concentrations of all three isotypes served as a standard for total IgA, IgG, and IgM levels. Standard curves were constructed using a computer program based on the four-parameter logistic model (Delta Soft, BioMetallics, Princeton, NJ). Sera from SCID mice that did not receive cells and normal BALB/c mice were used as controls.

Phenotype analysis

Peritoneal lymphocyte populations were analyzed by flow cytometry. A mAb against MHC class I (HLA-A, -B, and -C, Olympus Immunochemicals, Lake Success, NY) Ag was used to distinguish the human cells from the resident mouse cells. In addition, Abs against the surface pan-B (CD19) and -T cell markers (CD3, CD2, and CD5) were used to characterize the lymphocyte populations. All Abs were purchased from Becton Dickinson (San Jose, CA) unless stated otherwise.

Histology

Tissues were fixed in acid alcohol (95% ethanol, 5% glacial acetic acid), embedded in low-melting point paraffin, and processed according to the method of Sainte-Marie (24). For initial analysis, serial sections were stained with hematoxylin and eosin (H&E) or primary polyclonal Abs against human and mouse Ig (Southern Biotechnology Associates, Birmingham, AL). The T cell population was identified using mAb to CD2. To determine the isotypes of the Ig-containing cells, polyclonal Abs against human IgG, IgM, and IgA labeled with tetraethylrhodamine isothiocyanate (TRITC), FITC, or biotin (Southern Biotechnology), respectively, were incubated together on the same section. After an extensive wash in PBS, the secondary reagent, streptavidin-conjugated fluorochrome 7-amino-4-methyl-coumarin-3-acetate (AMCA) (Jackson), was added to visualize the biotinylated IgA Ab, yielding three-color immunofluorescence (25). Similar techniques were used to determine the isotype of cells containing cytoplasmic J chain and the presence of {kappa} and {lambda} light chains (Southern Biotechnology) (26). The polyclonal Abs were absorbed with mouse liver powder (Rockland, Gilbertsville, PA) to remove any nonspecific reactivity with murine B cells. The polyclonal Abs were shown to cross-react with other primate but not murine lymphocytes.

EBV status

Sections positive for primate Ig-containing cells were stained for the Epstein-Barr nuclear Ag (EBNA-1, Accurate Chemicals, Westbury, NY). The phenotype of EBV+ B cells was determined as described above.

Statistical analysis

All statistical analyses were performed with the InStat 2.0 (GraphPad, San Diego, CA) software package. Differences in the percentage of Ig-containing cells between human- and primate-engrafted SCID mice were determined by the Mann-Whitney test for nonparametric data.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The majority of human cells recovered from the peritoneal lavage are T cells and not B cells

An average of 2.4 x 106 cells (range: 0.5–6.5 x 106) was recovered from the peritoneal lavage. To determine the percentage of human lymphocytes, Abs against MHC class I Ags were used. Human lymphocytes represented an average of 44% (range: 19–86%) of the recovered peritoneal cells (Fig. 1GoA). T cells represented 41% (10–80%) of the peritoneal population, and less than 2% (0–6%) stained for the B cell surface Ag, CD19 (Fig. 1GoB). Phenotypes of the recovered PBMC were not altered by preincubation of the cells with any of the specified mitogens (Table IGo).



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  		FIGURE 1. Flow cytometric analysis of human cells recovered from peritoneal lavage. Representative data from two different mice are shown. A, Cells were stained with Abs against human MHC class I (HLA-A, -B, and -C) to distinguish the human cell population. B, In addition, the human T and B cell populations were analyzed by two-color flow cytometry using anti-human CD3 and CD19 Abs.

 
Primate B lineage cells are found in lymphocytic aggregates in the mesentery of engrafted SCID mice

H&E staining of the mesentery from reconstituted SCID mice revealed that lymphocytes were beneath the peritoneal lining, organized into microscopic, vascularized, and sometimes encapsulated aggregates that did not have a typical lymph node structure with germinal centers (Fig. 2GoA). Similar lymphocytic aggregates containing only IgM+ cells were found in the mesentery of normal BALB/c and C57BL/6 mice (Fig. 2GoB), but not in nonengrafted SCID mice. A second type of microscopic structure that appeared to be a small lymph node was also found in the mesentery in both engrafted (Fig. 2GoC) and nonengrafted SCID mice, as well as in normal mice (Fig. 2GoD). In normal animals, these structures contained mainly IgA+ (cytoplasmic and surface) and surface IgM+ cells, with few cytoplasmic IgG+ cells.



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  		FIGURE 2. A–D, Light microscopy of representative sections of H&E-stained mesentery structures in engrafted SCID mice and normal control mice. Histologic sections of lymphocytic aggregates in (A) hu-PBL-SCID mice and (B) C57BL/6 control mice. Histologic sections of small lymph nodes in the (C) hu-PBL-SCID and (D) BALB/c control mouse.

 
To determine whether these structures in the engrafted SCID mice contained human lymphocytes, sections were analyzed by immunofluorescence for the presence of human B or T cells. Ig+ lymphoblasts were found in the spleen and in aggregates located throughout the mesentery (Fig. 3GoA). In general, the small lymph nodes were devoid of human cells. By immunofluorescence staining, 82% of the mice were shown to be reconstituted with human cells of B lymphocyte lineage. Ig-containing cells were also found in 74% and 80% of the mice reconstituted with chimpanzee and pig-tailed macaque lymphocytes, respectively (Fig. 3Go, B and C). We did not observe any increased engraftment or change in phenotype of the human Ig-containing cells in studies comparing engraftment of freshly isolated PBMC to those of PBMC in association with HuGH or PBMC previously mitogen activated. Human T cells were found in the spleen but were rarely detected in the lymphoid aggregates.



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  		FIGURE 3. Immunofluorescence microscopy of lymphocytic aggregates. Tissue sections of mesentery stained with anti-human Ig-TRITC (red) from SCID mice engrafted with (A) human, (B) chimpanzee, and (C) pig-tailed macaque PBMC. D, To determine the clonality of the Ig-containing cells, sections were costained with polyclonal Abs against human {kappa}-FITC (green) and {lambda}-TRITC (red) light chains. Lymphocytic aggregates from the hu-PBL-SCID were analyzed by three-color immunofluorescence by staining with Abs to human (E) IgG-TRITC (red), (F) IgA-AMCA (blue), and (G) IgM-FITC (green) to determine the isotype of the Ig+ cells. H, To determine cytoplasmic J chain expression, sections were costained for anti-human IgG-FITC (green, left) and J chain-TRITC (red, right).

 
Phenotype of Ig-containing cells in the lymphocytic aggregates in the mesentery

Further characterization of the lymphocytic aggregates determined that the Ig-containing cells displayed morphologic features of lymphoblasts or plasma cells. Evaluation of light chain expression of the Ig-containing cells within the lymphoid aggregates revealed that 62% expressed {kappa} and 38% expressed {lambda} light chains (Fig. 3DGo). Analysis of the isotypes of the cytoplasmic Igs showed that IgG predominated and that IgA- and IgM-containing cells were generally found in approximately equal numbers (Table IIGo; Fig. 3Go, E, F, and G). There were no significant differences in isotype distribution of Ig-containing cells from mice engrafted with fresh or mitogen-stimulated PBMC from the same donor, or between mice engrafted with PBMC from different donors. Therefore, we combined the phenotypic data from the hu-PBL-SCID mice for comparisons with nonhuman primate-engrafted mice. Lymphocytic aggregates from SCID mice reconstituted with nonhuman primate PBMC were similar to lymphoid aggregate tissues from hu-PBL-SCID mice in that a greater percentage of the lymphoblasts contained cytoplasmic IgG. Significant differences in the percentages of IgG- and IgM-containing cells were observed in the hu-PBL-SCID mice when compared with the chimpanzee-PBL-SCID mice, but no significant differences were found between the mice engrafted with human and macaque lymphocytes, or between mice engrafted with macaque and chimpanzee lymphocytes (Table IIGo). In the hu-PBL-SCID mice, spleen sections showed repopulation only by IgM-containing cells, and aggregated lymphoblastic cells were found occasionally in the pleural cavity near the thymus. Interestingly, 69% of IgA-, 92% of IgM-, and 78% of IgG-containing cells expressed cytoplasmic J chain (Fig. 3GoH). We did not observe any significant repopulation of the gut, liver, thymus, lungs, or salivary glands by human or nonhuman primate B cells.


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  		Table II. Isotype of Ig-containing cells in lymphoid aggregates from SCID mice engrafted with primate PBMC

 
The majority of Ig-containing cells are not EBV+

To determine whether the human Ig-containing cells were infected with EBV, we stained the sections with Abs against the latent EBV nuclear Ag 1 (EBNA-1). At three weeks postinjection, less than 1% of the human lymphocytes were positive for EBNA-1 in the majority of tissues analyzed. In only two mice from the same donor did we find high numbers of EBNA-1+ cells.

Human Ig levels in serum, saliva, and bile

Human Igs were detected in sera and secretions of the reconstituted mice. Again, IgG was the predominant isotype found in the serum (range: 260-5000 µg/ml). Lower levels of IgM (range: 0.2–192 µg/ml) or IgA (3.7–331 µg/ml) were detected. Due to low sample volumes, only IgA was analyzed in bile and saliva samples. Human IgA was detected in the bile (54–477 ng/ml) of 5 of the 11 engrafted animals tested (45%) but was not detected in the saliva.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study we investigated the engraftment of human, chimpanzee, and pig-tailed macaque B lineage cells in SCID mice. To our knowledge, this is the first report on the phenotypes of B lineage cells from chimpanzee and pig-tailed macaque PBMC engrafted into SCID mice. Because we were interested in characterizing B cells in early engraftment, we evaluated various time points (1, 2, 3, 4, 7, 9, 14, and 21 days). Based on peritoneal cell recovery and levels of human Igs in mouse serum, most of the studies were performed 3 wk postinoculation. The engrafted primate cells were located in microscopic mesenteric lymphoid aggregates. These structures are present in immunocompetent mice and contain sIgM+ B cells. However, the phenotype of these cells in the engrafted SCID mice was characteristic of highly differentiated lymphoblasts compared with the sIg+ B cells present in these tissues in normal mice. In previous studies, cells that were removed from the peritoneal cavity produced anti-mouse Abs (27), suggesting that the increased differentiation state of the primate B cells is most likely due to a response to murine Ags. This finding may explain why in vitro activation of PBMC with mitogens before injection did not influence the phenotype or location of the engrafted primate B cells. Previous studies in hu-PBL-SCID mice have shown that engrafted B cells are capable of producing specific Abs to T cell-dependent Ags (20, 28, 29, 30, 31). Because T cells were rarely found within the lymphoid aggregates, it is still unclear if T cell-dependent events, such as cytokine-driven differentiation or activation of the lymphoblasts, resulting in the presence of an almost pure accumulation of polyclonal plasma cells, would be initiated within these aggregates or elsewhere in the peritoneum. At present, we do not know whether human T cells that mostly remain in the murine peritoneal cavity (this paper and 32 after i.p. injection of PBMC influence, albeit without direct contact, terminal B lymphocyte differentiation in these unique structures. Alternatively, endogenous murine cytokines may also be responsible for this effect.

Since most of the primate T cells were recovered from the peritoneal lavage or spleen, the state of activation of the T cell population may determine their localization in SCID mouse tissues. On the other hand, cells of B lineage were differentiated into lymphoblastoid or plasma cells, which characteristically lose CD19, thus explaining the decreased numbers of recovered CD19+ cells. In addition, the localization of B lymphoblasts into organized lymphoid structures may prevent their removal by peritoneal flushing. This localization may also explain why B lineage cells were not detected in the intestinal tissues. In other experiments in which mice were reconstituted with mouse (33, 34) or rat lymphocytes (H.H.D., unpublished data), B cells were detected in the intestinal lamina propria; in the SCID mice engrafted with rat lymphocytes, no mesenteric lymphocytic aggregates were found. Perhaps species differences in B cell homing receptors influence their tissue distribution.

T cells were not observed in the intestinal tissues. A previous report (8) suggested that T cells found in peripheral tissues, such as the spleen, had characteristics of activated cells, which down-regulate their homing receptors. This may explain the lack of T cell migration into the intestine.

The lymphoblasts found in the aggregates were polyclonal, as evidenced by expression of all three major Ig heavy chain and both light chain isotypes. The predominant class of Ig in the aggregates, as well as in serum, was IgG. IgA- and IgM-secreting cells comprised a higher percentage of the cells in the aggregates than the corresponding levels of these Igs in the serum. One explanation for this difference comes from studies that have shown the half-life of human serum IgA and IgM (36.9 and 23.1 h, respectively) to be much shorter than that of human IgG (11.9 days) in engrafted SCID mice (14). Our finding of IgA in the bile would indicate that there is transport of IgA from the circulation (35). An interesting observation was the expression of cytoplasmic J chain by the majority of Ig-containing cells, regardless of the isotype. This finding suggests that the B lineage cells are in a state of activation, typical of Ig- and J chain-producing cells (36, 37, 38). B lymphoblasts were found on occasion in the spleen and in the pleural fat, indicating that they can disseminate to other regions. These findings were consistent whether the mice were reconstituted with human, chimpanzee, or macaque PBMC, indicating that primate B cell engraftment is comparable among species.

Lymphomas that arise in hu-PBL-SCID mice have been shown to contain latent EBV Ags characteristic of EBV-infected cell lines (11, 39). Therefore, we examined the lymphocytic aggregates for the expression of EBV Ags. The majority of human lymphocytes were negative for EBV Ags at 3 weeks postinfection. This finding suggests that the phenotype of the Ig-containing cells in early engraftment of SCID mice is similar to that of mitogen-activated human peripheral blood lymphocytes (36) and not due to lymphoproliferative outgrowth of an EBV-transformed B cell. The lymphocytic tumors that are often described in engrafted SCID mice (10, 11, 18, 19, 39) may occur after long-term engraftment if the few EBV+ cells found in early engraftment become long-lived and invade areas such as the small lymph node-like structures, which are generally devoid of primate cells.

A possibility that the observed lymphoid structures, generated in SCID mice after the injection of human PBMC, may be analogous to the omental "milky spots" (also known as Milcheflecken or taches laiteuses) (40, 41, 42, 43, 44) has been considered. These structures are found with a high density in the omentum of many species, including humans and mice (41, 43). In mice and humans, the omental lymphoid structures contain self-replenishing lymphocytes of the B1 (CD5+) lineage and may serve as an additional site of B cell generation (45, 46, 47). However, the structural features of milky spots and the observed lymphoid aggregates described in this paper are different. The milky spots are found mainly in the omentum, are relatively small (difficult to see without magnification), contain mainly macrophages, T cells, B cells, and a few plasma cells, and are covered by porous epithelium, which allows the influx and efflux of cells (42, 44, 48, 49). In contrast, the lymphoid aggregates we observed in hu-PBL-SCID mice were found frequently in retroperitoneum (in the vicinity of the pancreas), hepatic hilus, and attached to organs such as the spleen. Typically, many of the aggregates were larger than putative milky spots, were encapsulated and contained fully differentiated plasma cells as the dominant population (Fig. 2Go, C and D, and Fig. 3GoD). Nevertheless, we cannot exclude the possibility that some of the structures observed (e.g., Fig. 2Go, A and B, and Fig. 3Go, E–G) are indeed milky spots induced in SCID mice by human PBMC. However, in contrast to mesenteric lymphoid structures of mice, which are known to populate other tissues with IgA plasma cell precursors, especially the intestines (45), we did not detect human plasma cells in significant numbers in the gut and other mucosal tissues of SCID mice. Thus, it appears that the cells remain in the aggregates without a marked dissemination into the periphery. Whether this is due to down-regulation of expression of mucosal or lymph node homing receptors on human B cells, as a consequence of their differentiation stage, is at present unknown. This does not seem to be the case with human T cells, which, especially after infection with HIV, migrate into the gut of SCID mice (this paper, and 32 .

Transfer of murine Peyer’s patch (PP) and peripheral and mesenteric lymph node cells (PBMC were not examined) into SCID mice resulted in repopulation of the immune system (50). However, there were some qualitative differences in repopulation of various host tissues depending on the source of the donor lymphoid cells. For example, transfer of PP cells yielded reconstitution of both lamina propria and intraepithelial cell compartments by IgA-producing plasma cells and T cells, while peripheral lymph node cells gave rise to T cells in lamina propria but not to IgA plasma cells (50). Because primate lymphoid cells from PP, spleen, and lymph nodes were unavailable for our experiments, we could not examine their repopulation potential and restricted our studies to PBMC.


   Acknowledgments
 
We thank Annette Pitts, Pam May, and Marion Spell for technical support. In addition, we would like to thank Dr. Russell Lindsey for his analysis of the histologic sections.


   Footnotes
 
1 This work was funded by National Institutes of Health Grants AI O7051, AI 28147, AI 23952, DE 12146, AI 32377, and CA 67386. Back

2 Present address: National Institutes of Dental Research, National Institutes of Health, 30 Convent Drive, Bethesda, MD 20892. Back

3 Address correspondence and reprint requests to Jiri Mestecky, Department of Microbiology, University of Alabama at Birmingham, 756 BBRB, 845 19th Street South, Birmingham, AL 35294. E-mail address: Back

4 Abbreviations used in this paper: HuGH, human growth hormone; H&E, hematoxylin and eosin; AMCA, 7-amino-4-methyl-coumarin-3-acetate; EBNA, Epstein-Barr nuclear Ag. Back

Received for publication January 16, 1998. Accepted for publication April 6, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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