Clostridioides difficile Binary Toxin Binding Component Increases Virulence in a Hamster Model.

Background: Clostridioides difficile is the leading cause of hospital-acquired gastrointestinal infection, in part due to the existence of binary toxin (CDT)-expressing hypervirulent strains. Although the effects of the CDT holotoxin on disease pathogenesis have been previously studied, we sought to investigate the role of the individual components of CDT during in vivo infection. Methods: To determine the contribution of the separate components of CDT during infection, we developed strains of C difficile expressing either CDTa or CDTb individually. We then infected both mice and hamsters with these novel mutant strains and monitored them for development of severe illness. Results: Although expression of CDTb without CDTa did not induce significant disease in a mouse model of C difficile infection, we found that complementation of a CDT-deficient C difficile strain with CDTb alone restored virulence in a hamster model of C difficile infection. Conclusions: Overall, this study demonstrates that the binding component of C difficile binary toxin, CDTb, contributes to virulence in a hamster model of infection.

Clostridioides difficile Binary Toxin Is Recognized by the Toll-Like Receptor 2/6 Heterodimer to Induce a Nuclear Factor-κB Response.

Clostridioides difficile infection (CDI) represents a significant burden on the health care system, one that is exacerbated by the emergence of binary toxin (CDT)-producing hypervirulent C. difficile strains. Previous work from our laboratory has shown that Toll-like receptor 2 (TLR2) recognizes CDT to induce inflammation. Here we explore the interactions of CDT with TLR2 and the impact on host immunity during CDI. We found that the TLR2/6 heterodimer, not TLR2/1, is responsible for CDT recognition, and that gene pathways including nuclear factor-κB and MAPK downstream of TLR2/6 are upregulated in mice with intact TLR2/6 signaling during CDI.

Type 2 cytokines IL-4 and IL-5 reduce severe outcomes from Clostridiodes difficile infection.

Clostridiodes difficile infection (CDI) is the leading cause of hospital-acquired gastrointestinal infections in the U.S. While the immune response to C. difficile is not well understood, it has been shown that severe disease is accompanied by high levels of infiltrating immune cells and pro-inflammatory cytokine production. This study tests the roles of two type 2 cytokines, IL-4 and IL-5, in mediating protection in a murine model of disease. Administration of IL-5 protected from mortality due to CDI, and both IL-4 and IL-5 were protective against severe disease symptoms. Together, the results from this study increase our understanding of how type 2 immune signaling processes are protective from severe C. difficile infection.

TLR2 as a Therapeutic Target in Bacterial Infection.

Toll-like receptor (TLR) 2 recognizes and responds to threats early in bacterial infections and can influence the downstream immune response to the host's benefit or detriment. Therapeutic modulation of TLR2 signaling represents an underutilized opportunity to moderate the immune response to infection to promote an improved outcome for the host.

Gut microbiome communication with bone marrow regulates susceptibility to amebiasis.

The microbiome provides resistance to infection. However, the underlying mechanisms are poorly understood. We demonstrate that colonization with the intestinal bacterium Clostridium scindens protects from Entamoeba histolytica colitis via innate immunity. Introduction of C. scindens into the gut microbiota epigenetically altered and expanded bone marrow granulocyte-monocyte progenitors (GMPs) and resulted in increased intestinal neutrophils with subsequent challenge with E. histolytica. Introduction of C. scindens alone was sufficient to expand GMPs in gnotobiotic mice. Adoptive transfer of bone marrow from C. scindens-colonized mice into naive mice protected against amebic colitis and increased intestinal neutrophils. Children without E. histolytica diarrhea also had a higher abundance of Lachnoclostridia. Lachnoclostridia C. scindens can metabolize the bile salt cholate, so we measured deoxycholate and discovered that it was increased in the sera of C. scindens-colonized specific pathogen-free and gnotobiotic mice, as well as in children protected from amebiasis. Administration of deoxycholate alone increased GMPs and provided protection from amebiasis. We elucidated a mechanism by which C. scindens and the microbially metabolized bile salt deoxycholic acid alter hematopoietic precursors and provide innate protection from later infection with E. histolytica.

IL-33 drives group 2 innate lymphoid cell-mediated protection during Clostridium difficile infection.

Clostridium difficile (C. difficile) incidence has tripled over the past 15 years and is attributed to the emergence of hypervirulent strains. While it is clear that C. difficile toxins cause damaging colonic inflammation, the immune mechanisms protecting from tissue damage require further investigation. Through a transcriptome analysis, we identify IL-33 as an immune target upregulated in response to hypervirulent C. difficile. We demonstrate that IL-33 prevents C. difficile-associated mortality and epithelial disruption independently of bacterial burden or toxin expression. IL-33 drives colonic group 2 innate lymphoid cell (ILC2) activation during infection and IL-33 activated ILC2s are sufficient to prevent disease. Furthermore, intestinal IL-33 expression is regulated by the microbiota as fecal microbiota transplantation (FMT) rescues antibiotic-associated depletion of IL-33. Lastly, dysregulated IL-33 signaling via the decoy receptor, sST2, predicts C. difficile-associated mortality in human patients. Thus, IL-33 signaling to ILC2s is an important mechanism of defense from C. difficile colitis.

Colitis-Induced Th17 Cells Increase the Risk for Severe Subsequent Clostridium difficile Infection.

Clostridium difficile infection (CDI) is the number one hospital-acquired infection in the United States. CDI is more common and severe in inflammatory bowel disease patients. Here, we studied the mechanism by which prior colitis exacerbates CDI. Mice were given dextran sulfate sodium (DSS) colitis, recovered for 2 weeks, and then were infected with C. difficile. Mortality and CDI severity were increased in DSS-treated mice compared to controls. Severe CDI is dependent on CD4+ T cells, which persist after colitis-associated inflammation subsides. Adoptive transfer of Th17 cells to naive mice is sufficient to increase CDI-associated mortality through elevated IL-17 production. Finally, in humans, the Th17 cytokines IL-6 and IL-23 associate with severe CDI, and patients with high serum IL-6 are 7.6 times more likely to die post infection. These findings establish a central role for Th17 cells in CDI pathogenesis following colitis and identify them as a potential target for preventing severe disease.

Biological Studies and Target Engagement of the 2- C-Methyl-d-Erythritol 4-Phosphate Cytidylyltransferase (IspD)-Targeting Antimalarial Agent (1 R,3 S)-MMV008138 and Analogs.

Malaria continues to be one of the deadliest diseases worldwide, and the emergence of drug resistance parasites is a constant threat. Plasmodium parasites utilize the methylerythritol phosphate (MEP) pathway to synthesize isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), which are essential for parasite growth. Previously, we and others identified that the Malaria Box compound MMV008138 targets the apicoplast and that parasite growth inhibition by this compound can be reversed by supplementation of IPP. Further work has revealed that MMV008138 targets the enzyme 2- C-methyl-d-erythritol 4-phosphate cytidylyltransferase (IspD) in the MEP pathway, which converts MEP and cytidine triphosphate (CTP) to cytidinediphosphate methylerythritol (CDP-ME) and pyrophosphate. In this work, we sought to gain insight into the structure-activity relationships by probing the ability of MMV008138 analogs to inhibit PfIspD recombinant enzyme. Here, we report PfIspD inhibition data for fosmidomycin (FOS) and 19 previously disclosed analogs and report parasite growth and PfIspD inhibition data for 27 new analogs of MMV008138. In addition, we show that MMV008138 does not target the recently characterized human IspD, reinforcing MMV008138 as a prototype of a new class of species-selective IspD-targeting antimalarial agents.

Determination of the active stereoisomer of the MEP pathway-targeting antimalarial agent MMV008138, and initial structure-activity studies.

Compounds that target isoprenoid biosynthesis in Plasmodium falciparum could be a welcome addition to malaria chemotherapy, since the methylerythritol phosphate (MEP) pathway used by the parasite is not present in humans. We previously reported that MMV008138 targets the apicoplast of P. falciparum and that its target in the MEP pathway differs from that of Fosmidomycin. In this Letter, we determine that the active stereoisomer of MMV008138 is 4a, which is (1R,3S)-configured. 2',4'-Disubstitution of the D ring was also found to be crucial for inhibition of the parasite growth. Limited variation of the C3-carboxylic acid substituent was carried out, and methylamide derivative 8a was found to be more potent than 4a; other amides, acylhydrazines, and esters were less potent. Finally, lead compounds 4a, 4e, 4f, 4h, 8a, and 8e did not inhibit growth of Escherichia coli, suggesting that protozoan-selective inhibition of the MEP pathway of P. falciparum can be achieved.

The immune response of foetal calves.

The immunological response of foetal calves to tetanus toxoid was investigated. Emphasis was placed on foetal immunocompetence and how this related to responses seen in adult cattle. The establishment of indwelling cannulas in the efferent prescapular lymphatic ducts and superficial veins of foetal calves allowed continual monitoring of cellular and humoral changes in efferent lymph and peripheral blood. Foetal calves from 195 to 253 days gestational age had the capacity to mount cell-mediated and humoral immune responses of similar character and magnitude as adult cattle to tetanus toxoid. Intravenous and subcutaneous routes of challenge with tetanus toxoid resulted in specific antibody production which peaked 26 to 31 days after vaccination. Significant tetanus toxoid-stimulated lymphocyte proliferation was present 4 to 6 weeks after vaccination with tetanus toxoid in both a foetus and an adult. After antigenic challenge lymphocytes remained the predominant cell type in efferent prescapular lymph of foetuses and cows while at the same time a marked shift to the left, characterised by band neutrophils and neutrophilic metamyelocytes occurred in peripheral blood. Lymph flow rate increased and cell concentration decreased after antigenic challenge.

The cellular response of lambs to Brucella ovis before and after birth.

The immunological response of lambs to Brucella ovis before and after birth was investigated. The establishment of indwelling cannulas in the efferent prescapular lymphatic ducts of foetal lambs allowed continual monitoring of the immune response of a single lymph node. Foetal lambs in the last trimester of pregnancy were shown to mount a strong cell-mediated immune response to B. ovis. Lymphocytes from the challenged lymph node stimulated with B. ovis in vitro usually first reacted significantly and had highest [3H]-thymidine incorporation between 4 and 6 days after primary and secondary challenge, whereas, lymphocytes from the unchallenged node did not exhibit significant [3H]-thymidine incorporation until some 24 h later. Lymphocytes from these lambs challenged as foetuses still exhibited significant [3H]-thymidine incorporation in response to B. ovis for 4 to 5 months after birth. The proportion of surface immunoglobulin-positive cells in efferent prescapular lymph of unchallenged lambs ranged from 0.5 to 2.0% but after B. ovis challenge this proportion ranged from 2.7 to 8.7% between 4 to 6 days after challenge. By 9 to 12 days after challenge, the proportion had declined to pre-challenge values.

The proliferative responses of lymphocytes from foetal calves and adult cattle.

This paper describes the proliferative responses of prescapular lymph lymphocytes and peripheral blood lymphocytes of foetal calves compared with cells of similar origin from adult cattle. Lymph lymphocytes were collected continuously by means of cannulation of efferent lymphatic ducts of the prescapular lymph node of foetal calves and adult cattle. Peripheral blood lymphocytes were collected from the foetus by means of cannulation of superficial veins of the foetus or of the umbilical vessels and from the jugular vein of adults. Foetal lymphocytes in one-way mixed lymphocyte culture stimulated and responded as well as adult lymphocytes. Foetal cells stimulated and responded more to cells from unrelated animals than to cells from their dam. Lymph lymphocytes from foetal calves between 188 and 253 days of gestation proliferated as well as adult lymphocytes and at a high level after stimulation with concanavalin A, phytohaemagglutinin and pokeweed mitogen. Response to stimulation with lipopolysaccharide, soybean agglutinin and wheat-germ agglutinin was variable but generally low and within the same range recorded by adult cells. Proliferation by foetal and adult whole-blood cultures was on occasions as high as that recorded by separated lymphocytes, even though fewer lymphocytes were initially present in the whole-blood cultures. Foetal lymph lymphocytes exhibited lower proliferative responses in autologous lymph plasma than in foetal calf serum or pooled foetal lymph plasma. There was no consistent depression of proliferation by culture medium supplements from pregnant animals. Rabbit serum consistently abrogated responses. Fetuin at final concentrations of greater than 2.5 mg/ml significantly depressed proliferation in foetal and adult lymphocytes from efferent lymph and peripheral blood.

Fetal lambs are depleted of IgM+ cells following a single injection of an anti-IgM antibody early in gestation.

B-cell depleted fetal sheep were created following a single injection of an anti-IgM monoclonal antibody early in gestation. Six sheep fetuses were given a single intraperitoneal injection of a monoclonal antibody directed against IgM at 63 days of gestation (gestation in sheep = 150 days). The fetuses were killed at 138-142 days of gestation and lymphoid tissues were collected for subsequent light microscopy and immunohistochemical examination. The ileal and jejunal Peyer's patch (PP) follicles in four of the six injected fetuses were markedly reduced in size. Cells in the rudimentary follicles of the ileal PP of these animals showed no reactivity for IgM and most were negative for CD45. The dome regions contained many T cells, which were predominantly CD8+ cells and included gamma delta T cells. The interfollicular areas of the PP of the markedly affected fetuses contained large populations of T cells. The spleen and lymph nodes were also markedly depleted of IgM+ cells and these tissues contained only a small, scattered population of weakly IgM+ cells. Follicular accumulations of IgM+ cells were absent. Large populations of T cells were present in the white pulp of the spleen and cortex of the lymph nodes. The liver did not contain IgM+ cells and the medulla of the thymus was depleted of IgM+ cells. The results of this study suggest that a surface IgM+ B-cell population is present in the sheep fetus at 63 days of gestation, which is essential for the colonization of the ileal PP and subsequent B-cell development.

Ontogeny of milky spots in the fetal lamb omentum.

Milky spots in the fetal lamb omentum were observed by light and electron microscopy under normal conditions and after intraperitoneal carbon injection in utero. Rudimentary milky spots first appeared as small aggregations of cells along capillaries of the omental branch of the right ruminal artery at 72 days of gestation. At 116 days of gestation, macrophages were detected immunohistochemically in the milky spots. At 125 days of gestation, T cells were detected in the milky spots, but B cells were absent. Under the conditions induced by intraperitoneal injection of carbon suspension, openings were observed between the omental mesothelial cells, and macrophage aggregations appeared on the surface of the omentum through the openings. At 148 days of gestation (newborn), the milky spots were noted as black spots because of aggregation of carbon laden macrophages. The present study demonstrates that milky spots are present at fixed sites in the fetal lamb omentum by the middle of the term, and that at birth the macrophages on the spots already possess phagocytotic ability. The fetus develops the ability to protect the peritoneal cavity by producing peritoneal macrophages from the milky spots in the greater omentum even in utero.

Retarded and excessive development of skin appendages in fetal lambs in response to thyroidectomy before wool follicle appearance.

The impact on wool follicles of development in an athyroid environment was studied in a series of twin fetal lambs by surgically thyroidectomizing one of each pair before the appearance of follicle buds and comparing development of epidermal appendages in it with their development in the normal co-twin. Thyroidectomy was undertaken at 51 to 54 days' gestation, i.e. after approximately one-third of the gestation period. Each treated fetus was then replaced in the uterus, allowing pregnancy to continue. Eight pairs of twins were removed at intervals from 67 to 122 days' gestation and skin samples from the thyroidectomized and the intact twins were compared. Micromorphometric examination of the samples was used to assess quantitatively the effects of thyroid deprivation on wool follicle development. In thyroidectomized fetuses there was a failure of keratinization in primary wool follicles, an absence of secondary follicles, a tendency to excessive follicular branching and sweat gland development, and a paucity of sebaceous gland formation. The density of wool follicles was substantially increased, but the mean cross-sectional area of these follicles was reduced. The effects of very early thyroidectomy imply that the thyroid plays a role in the stimulation and regulation of wool follicle differentiation. To test the reversibility of the effects observed in the skin of thyroidectomized fetuses, grafts from these animals were transplanted to normal, young fetal lambs. Subsequent examination of grafted skin revealed that complete keratinization had occurred but that none of the other abnormal features had been reversed.

Morpho-physiological function and role of omental milky spots as omentum-associated lymphoid tissue (OALT) in the peritoneal cavity.

The morpho-physiological function and role of milky spots in the greater omentum are reviewed. These milky spots are composed of cellular aggregations of mesenchymal cells, mainly macrophages and lymphocytes, surrounding capillary convolutions termed omental glomeruli. Initial lymphatics of the omentum begin at the milky spots and drain into lymph collectors. The lymphatic capillaries in the omental milky spots take part in the absorption of various substances from the peritoneal cavity. Omental milky spots probably act as the first line of defense in the peritoneal cavity and therefore are immunologically important. In human infants, most of the cells in these milky spots are macrophages (49%); less common are B lymphocytes (29%) and T lymphocytes (12%). Whereas macrophages form clusters near the peritoneal surface of the milky spots and are oriented toward the peritoneal cavity for migration, clusters of B and T lymphocytes are typically found in periarteriolar locations within the milky spots. This cell zonation facilitates phagocytosis and processing of circulating antigens and foreign bodies which emanate from the peritoneal cavity. During inflammation, the number and size of omental milky spots dramatically increase, and some develop germinal centers within the lymphatic follicles and produce antibodies. During intraperitoneal immunotherapy, the omental milky spots and their cellular elements may be activated by intraperitoneal administration of biological response modifiers, and thereby represent an important immunoregulatory system for the peritoneal cavity. Omental milky spots are also closely linked to the dissemination of cancer cells. Thus, intraperitoneally inoculated experimental tumor cells selectively invade the milky spots and proliferate there to form tumor nodules. This occurrence is relevant to clinical practice where nodular metastases to the omentum are common. Omental milky spots are analogous to regional lymph nodes and as such are the omentum-associated lymphoid tissues and participate in intraperitoneal immune reactions.

Technique for in situ excision of distended samples of greater omentum from small laboratory animals.

A simple and reliable technique is described for in situ excision of distended samples of greater omentum from small laboratory animals. The omental bursa is distended by injecting whipped hen egg white. Filter paper frames then are applied to the selected areas of distended omentum and samples of omental membrane are excised together with the filter paper frames. This sampling technique yields undamaged materials suitable for various research purposes.

Activation of omental milky spots and milky spot macrophages by intraperitoneal administration of a streptococcal preparation, OK-432.

Omental milky spots are omentum-associated lymphoid tissues that cede peritoneal macrophages and participate in the immunity of the peritoneal cavity. We studied the changing surface features of milky spots and milky spot macrophages of Wistar rats, following the i.p. administration of OK-432, a killed streptococcal preparation (1 Klinische Einheit (unit) in 5 ml of phosphate-buffered saline) by the use of scanning electron microscopy. OK-432-activated macrophages demonstrated marked surface membrane activity and migrated through the stomata of the milky spot into the peritoneal cavity. The characteristic features of activated milky spots and milky spot macrophages were noted as early as 3 h following the administration of OK-432, and continued to be observed until 7 days after the injection. By 14 days after the injection, the structural integrity of the milky spot was partially lost. The activation of milky spots and milky spot macrophages by OK-432 provides a convenient in vivo system for the monitoring and study of i.p. cellular events.

Major lymph nodes of the head of the fallow deer (Dama dama) and lymphatic drainage of antlers.

Heads from 10 fallow deer bucks were examined to provide a description of the major lymph nodes in this region. The distribution and size of these nodes were similar to those of sheep and goats. To determine whether there was drainage of lymph from the skin of antlers, and to follow the route of this drainage, a solution containing Evans blue was injected intradermally into the antlers of 2 bucks whilst the animals were anaesthetised. Dye appeared in tracheal lymph ducts 14 to 30 min after injection. The spread of blue colouration in lymphatic ducts and nodes, seen at post-mortem examination, indicated that lymph flowed from antlers laterally into the ipsilateral parotid lymph nodes and from these via medial and lateral retropharyngeal lymph nodes to the tracheal ducts.