James A. Harp

Immunity in The Mammary Gland

The ruminant mammary gland is an extremely important economic organ in that it provides a major nutrition source for a significant portion of the worlds human population. The ruminant mammary gland is also responsible for providing protective immunity to neonates and for defending itself from invading pathogens. A wide array of humoral and cellular immune mechanisms are present in the mammary gland and actively participate in providing immunity to newborns and the mammary gland per se. The acute inflammatory response is essential in determining the outcome of intramammary challenge, and factors affecting innate and adaptive immunity in the context of mammary health are reviewed in detail. The ruminant mammary gland is also unique in that lymphocyte trafficking, which is essential to adaptive immunity, is shared with the peripheral immune system rather than the common mucosal immune system.

In vitro proliferation and production of gamma interferon by murine CD4+ cells in response to Cryptosporidium parvum antigen

Spleen cells from mice immunized with Cryptosporidium parvum were enriched for T cells by passage over an affinity chromatography column. The proliferative response of these cells was > 2-fold higher than the response of unenriched cells. T-enriched cells were enriched further for either CD4+ cells or CD8+ cells. The proliferative response of CD4-enriched cells was > 4-fold higher than the response by unenriched cells. CD8+ cells were essentially nonresponsive to C. parvum antigen. Culture supernatant fractions from these variously enriched splenocyte populations were assayed for cytokine production. Cultures containing CD4+ cells produced gamma interferon and interleukin-2 following incubation with C. parvum antigen. None of the cultures produced interleukin-4. Production of gamma interferon and interleukin-2, but not interleukin-4, is characteristic of the previously described Th1 helper cell subset. Our data indicate that a subset of murine lymphocytes consistent with the Th1 helper cell phenotype proliferates following in vitro stimulation with C. parvum antigen.

Protection of calves with a vaccine against Cryptosporidium parvum.

Cryptosporidium parvum causes enteric infection and diarrhea in calves, other species of economically important livestock, and humans. There are no effective treatments currently licensed for this parasite, and preventive measures are difficult. In addition to direct economic losses to the cattle industry, infected calves may contaminate water supplies with oocysts and contribute to human cryptosporidiosis. We have developed a vaccine offering partial protection against C. parvum infection in calves. Nine calves received an oral preparation of lyophilized C. parvum oocysts shortly after birth, and 10 calves served as nonvaccinated controls. All calves received colostrum. At 1 wk of age, all calves were administered 10(4) viable C. parvum oocysts orally. Clinical disease and oocyst shedding were monitored daily. Mean duration of diarrhea was 4 days for control calves and 1.7 days for vaccinated calves. Mean duration of oocyst shedding was 5.3 days for control calves and 2 days for vaccinated calves. These differences were statistically significant and suggest that this vaccine has the potential to reduce diarrhea and oocyst shedding caused by C. parvum.

Numbers and percent of T lymphocytes in bovine peripheral blood during the periparturient period

To determine if periparturient immunosuppression in dairy cattle might be due to an alteration in total numbers of percent of T lymphocytes, we examined the numbers and percent of T lymphocyte subsets in peripheral blood from periparturient dairy cows, some of which received recombinant bovine granulocyte colony stimulating factor (rbG-CSF) during the study. Beginning 2 weeks preparatum through 4 weeks postpartum, peripheral blood mononuclear cells (PBMC) were collected and labeled with monoclonal antibodies to BoCD5, BoCD4, and BoCD8, and the percent of cells positive for each marker measured by flow cytometry. The percent of PBMC expressing BoCD5 (total T cells), and BoCD8 (T suppressor/cytotoxic cells) was not significantly different between the groups, or at different times before and after calving. The percent of PBMC expressing BoCD4 (T helper cells) was not significantly different between the groups, however, within both groups there was a higher percent of BoCD4+ cells after calving than during the prepartum period. In cows receiving rbG-CSF, total numbers of PBMC were significantly increased compared to controls during the postpartum treatment period.

Regulation of mitogenic responses by bovine milk leukocytes.

Bovine milk lymphocytes are less responsive to in vitro mitogen stimulation than peripheral blood lymphocytes (PBL). In this study, milk leukocytes (ML) or their soluble products, were co-cultured with mitogen stimulated PBL to determine if suppression could be transferred to normally responsive cells. Addition of either ML (treated with mitomycin C to prevent cell division), or supernatant from ML cultures to cultures of autologous PBL resulted in a reduction of mitogenesis by the PBL, but no suppression was seen with addition of treated PBL or PBL supernatant. Suppression was greater when the ML were from animals with chronic staphylococcal infection. Suppression by ML supernatant was not due to toxicity to the responders, since addition at the latter stages of culture had no effect on the response. These results indicate that reduced mitogenesis by milk lymphocytes may be due to the presence of suppressor cells or molecules.

Treatment with Neurokinin-1 Receptor Antagonist Reduces Severity of Inflammatory Bowel Disease Induced by Cryptosporidium parvum

ABSTRACT Inflammatory bowel disease (IBD) is a chronic, debilitating disorder of uncertain and perhaps multiple etiologies. It is believed to be due in part to disregulation of the immune system. Neuroimmune interactions may be involved in induction or maintenance of IBD. In the present study, we examined the potential role of a neurotransmitter, substance P, in a mouse model of IBD. We found that binding sites for substance P, and more specifically, neurokinin-1 receptors, were upregulated in intestinal tissue of mice with IBD-like syndrome. Dosing of mice with LY303870, a neurokinin-1 receptor antagonist, reduced the severity of IBD, and treatment of mice with preexisting IBD allowed partial healing of lesions. We hypothesize that blocking the binding of substance P to the neurokinin-1 receptor interrupts the inflammatory cascade that triggers and maintains intestinal lesions of IBD.

Lymphocyte recirculation in cattle: Patterns of localization by mammary and mesenteric lymph node lymphocytes

We examined patterns of lymphocyte localization in female dairy cattle following infusion of 51Cr-labeled autologous lymphocytes prepared from surgically excised mammary or ileal mesenteric lymph nodes. Labeled lymphocytes prepared from mammary lymph nodes were recovered in proportionally high numbers from mammary and prescapular lymph nodes, and in low numbers from intestinal mesenteric nodes. This pattern was observed in both heifers and lactating cows. In contrast, labeled lymphocytes prepared from ileal mesenteric lymph nodes of lactating cows were recovered in proportionally high numbers from intestinal mesenteric nodes, and in low numbers from mammary and prescapular nodes. These findings, when compared with previous results in sheep and swine, support the hypothesis that lymphocytes do not migrate efficiently between the gut and mammary gland of ruminants.

[1N,12N]Bis(Ethyl)-cis-6,7-Dehydrospermine: a New Drug for Treatment and Prevention of Cryptosporidium parvum Infection of Mice Deficient in T-Cell Receptor Alpha

ABSTRACT Cryptosporidium parvum infection of T-cell receptor alpha (TCR-α)-deficient mice results in a persistent infection. In this study, treatment with a polyamine analogue (SL-11047) preventedC. parvum infection in suckling TCR-α-deficient mice and cleared an existing infection in older mice. Treatment with putrescine, while capable of preventing infection, did not clear C. parvum from previously infected mice. These findings provide further evidence that polyamine metabolic pathways are targets for new anticryptosporidial chemotherapeutic agents.

In Vitro Murine Lymphocyte Blastogenic Responses to Cryptosporidium parvum

Spleen and mesenteric lymph node lymphocytes from both Cryptosporidium parvum-exposed and unexposed mice were cultured with antigen (Ag) prepared from C. parvum oocysts. Spleen lymphocytes from oral-, intraperitoneal-, or oral + intraperitoneal-exposed mice did not respond significantly (P greater than 0.05) to Ag stimulation. Spleen lymphocytes from multioral-exposed mice, however, demonstrated significant (P less than or equal to 0.01) Ag-specific blastogenesis. Mesenteric lymph node lymphocytes did not respond to in vitro Ag stimulation regardless of the route of in vivo priming. These results demonstrate an in vitro cell-mediated immune response against C. parvum by lymphocytes in murine spleen.

Lymphocyte localization in lymph nodes of pubescent, prepartum, and postpartum sheep

It is generally accepted that lymphocytes associated with the mammary mucosal immune system of non-ruminants may be largely derived from gut-associated lymphoid tissue (GALT). The relationship between the mammary immune system and the GALT of ruminants has not been clearly defined. To address this question, we examined patterns of lymphocyte localization in sheep by 51Cr-labeled lymphocytes following infusion back into donor ewes. We found that lymphocytes taken from mammary lymph nodes of pubescent ewes returned preferentially to mammary nodes, while in prepartum and postpartum ewes, mammary node cells localized equally well in mammary and mesenteric lymph nodes. In contrast, ileal mesenteric lymph node cells from pubescent ewes localized equally well in mammary and mesenteric nodes, but in prepartum and postpartum ewes, localization in mammary nodes was markedly reduced. Comparison of the homing patterns of mammary, mesenteric, and peripheral lymph node cells indicated that mammary node cells behaved similarly to peripheral, rather than mesenteric node cells. This information may be relevant to the extent of communication between the gut and mammary gland in ruminants.