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The mechanisms of efficacy for fecal microbiota# transplantation (FMT) in treating recurrent Clostridioides difficile infection (rCDI) remain poorly defined, with restored gut microbiota-bile acid interactions representing one possible explanation. Furthermore, the potential implications for host physiology of these FMT-related changes in gut bile acid metabolism are also not well explored. In this study, we investigated the impact of FMT for rCDI upon signalling through the farnesoid X receptor (FXR)-fibroblast growth factor (FGF) pathway. Herein, we identify that in addition to restoration of gut microbiota and bile acid profiles, FMT for rCDI is accompanied by a significant, sustained increase in circulating levels of FGF19 and reduction in FGF21. These FGF changes were associated with weight gain post-FMT, to a level not exceeding the pre-rCDI baseline. Collectively, these data support the hypothesis that the restoration of gut microbial communities by FMT for rCDI is associated with an upregulated FXR-FGF pathway, and highlight the potential systemic effect of FMT.

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-C. difficile antibody levels in human sera and in separate preparations of polyclonal IVIg. The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. Herein, a combination of isotype (IgG, IgA IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), precursor form of B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027) and control proteins (Tetanus toxoid and Candida albicans) were determined by protein microarray. Microarrays were probed with sera from individuals with C. difficile infection (CDI), cystic fibrosis (CF) without diarrhea, healthy controls and individuals pre- and post-IVIg therapy for treatment of CDI, combined immunodeficiency disorder and chronic inflammatory demyelinating polyradiculopathy. Significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels were seen between patient groups, commercial preparations of IVIg and sera before and following IVIg administration. A significant correlation was observed between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C. difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, it is anticipated that microarray technology may help optimize and select the most clinicall

Orbital inflammatory disease is a rare ophthalmic manifestation of Crohn’s disease. Inflammation is characteristically non-specific, involving one or multiple structures of the orbit. Mechanisms of disease and optimal methods of treatment are poorly understood. The aim of this report is to present three cases of orbital involvement in Crohn’s disease. A retrospective case note review of patients with orbital inflammatory disease and Crohn’s disease was performed at our academic centre to determine the clinical, imaging and histopathological features of this condition and its relationship to intestinal Crohn’s disease. Three patients were identified with orbital inflammatory manifestations complicating Crohn’s disease. All patients described were female with active intestinal disease and had a history of treatment with immunosuppressive therapies. Similarities were observed in clinical presentations with variance noted in radiological and histopathological findings. In all cases, symptoms improved with oral corticosteroids or non-steroidal drugs in combination with anti-tumour necrosis factor (anti-TNF) agents. Inflammatory bowel disease-related orbital complications are rare but potentially vision-threatening. It is important to consider mimics of orbital inflammatory disease such as systemic inflammatory disease, malignancy, congenital malformations, infection and trauma when formulating a comprehensive differential diagnosis. Therapeutic intervention is directed towards preservation of vision and orbital function and reducing the acute inflammatory process. Corticosteroids are typically the initial treatment of choice for moderate-to-severe disease, although several classes of immunomodulatory agents have been variably useful in treating this condition. Heightened awareness and close cooperation between gastroenterologists and ophthalmologists is mandatory.

The prevalence of serum antibodies against Clostridium difficile (CD) toxins A and B in healthy populations have prompted interest in evaluating the therapeutic activity of intravenous immunoglobulin (IVIg) in individuals experiencing severe or recurrent C. difficile infection (CDI). Despite some promising case reports, a definitive clinical role for IVIg in CDI remains unclear. Contradictory results may be attributed to a lack of consensus regarding optimal dose, timing of administration and patient selection as well as variability in specific antibody content between commercial preparations. The purpose of this study was to investigate retrospectively the efficacy of three commercial preparations of IVIg for treating severe or recurrent CDI. In subsequent mechanistic studies using protein microarray and toxin neutralization assays, all IVIg preparations were analysed for specific binding and neutralizing antibodies (NAb) to CD antigens in vitro and the presence of anti-toxin NAbs in vivo following IVIg infusion. A therapeutic response to IVIg was observed in 41% (10 of 17) of the CDI patients. Significant variability in multi-isotype specific antibodies to a 7-plex panel of CD antigens and toxin neutralization efficacies were observed between IVIg preparations and also in patient sera before and after IVIg administration. These results extend our current understanding of population immunity to CD and support the inclusion of surface layer proteins and binary toxin antigens in CD vaccines. Future strategies could enhance IVIg treatment response rates by using protein microarray to preselect donor plasma/serum with the highest levels of anti-CD antibodies and/or anti-toxin neutralizing capacities prior to fractionation.

Objectives: Despite multiple risk factors and a high rate of colonization for Clostridium difficile, the occurrence of C. difficile infection in patients with cystic fibrosis is rare. The aim of this study was to compare the prevalence of binding C. difficile toxin-specific immunoglobulin (Ig)A, IgG and anti-toxin neutralizing antibodies in the sera of adults with cystic fibrosis, symptomatic C. difficile infection (without cystic fibrosis) and healthy controls. Methods: Subclass-specific IgA and IgG responses to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B only expressing strain (CCUG 20309) and precursor form of B fragment of binary toxin, pCDTb, were determined by protein microarray. Neutralizing antibodies to C. difficile toxins A and B were evaluated using a Caco-2 cell-based neutralization assay. Results: Serum IgA anti-toxin A and B levels and neutralizing antibodies against toxin A were significantly higher in adult cystic fibrosis patients (n=16) compared with healthy controls (n=17) and patients with symptomatic C. difficile infection (n=16); p≤0.05. The same pattern of response prevailed for IgG, except that there was no difference in anti-toxin A IgG levels between the groups. Compared with healthy controls (toxins A and B) and patients with C. difficile infection (toxin A), sera from cystic fibrosis patients exhibited significantly stronger protective anti-toxin neutralizing antibody responses. Conclusion: A superior ability to generate robust humoral immunity to C. difficile toxins in the cystic fibrosis population is likely to confer protection against symptomatic C. difficile infection. This protection may be lost in the post-transplantation setting, where sera-monitoring of anti-C. difficile toxin antibody titers may be of clinical value.

Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotypespecific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated<10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost.

Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotypespecific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated<10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost.

C. difficile infection (CDI) is rarely reported in cystic fibrosis (CF) patients despite frequent hospitalisations and antibiotic usage. Conversely, the prevalence of CDI in inflammatory bowel disease (IBD) has received increased attention. We investigated components of the IgG-specific humoral immune response to C. difficile toxins A and B in patients with C. difficile-associated diarrhoea (CDAD), IBD patients with CDI, CF patients and healthy controls. Serum anti-toxin IgG was determined by ELISA. Circulating antigen-activated B-cells were investigated using Alexa Fluor 488-labelled toxin A and assessed by flow cytometry. Following induction of differentiation of memory B-cells, toxin A- and B-specific antibody secreting cells (ASCs) were quantified using ELISpot. We present the first data showing levels of serum anti-toxin A and B antibodies were significantly higher in patients with CF (without a history of CDI) than in CDAD patients and were stably maintained over time. Notably, the CDAD patients were significantly older than the CF patients. We also show that circulating toxin A-specific memory B-cells (IgD-negative) can be detected in CDAD patients [0.92 (0.09–1.78)%], and were prominent (5.64%, 1.14%) in two CF patients who were asymptomatic carriers of C. difficile. There was correlation between toxin A- and B-specific ASCs, with significantly higher proportions of the latter seen. In some with CDAD, high serum antibody levels were seen to only one of the two toxins. Mucosal secretion of toxin-specific IgG was detected in an additional group of IBD patients with no history of CDI. We conclude that enhanced and stable humoral immune responses to toxins A and B may protect CF and some IBD patients against CDI. The impaired ability to generate strong and/or sustained toxin-specific antibody and memory B-cell responses may increase susceptibility

The mechanisms of efficacy for fecal microbiota# transplantation (FMT) in treating recurrent Clostridioides difficile infection (rCDI) remain poorly defined, with restored gut microbiota-bile acid interactions representing one possible explanation. Furthermore, the potential implications for host physiology of these FMT-related changes in gut bile acid metabolism are also not well explored. In this study, we investigated the impact of FMT for rCDI upon signalling through the farnesoid X receptor (FXR)-fibroblast growth factor (FGF) pathway. Herein, we identify that in addition to restoration of gut microbiota and bile acid profiles, FMT for rCDI is accompanied by a significant, sustained increase in circulating levels of FGF19 and reduction in FGF21. These FGF changes were associated with weight gain post-FMT, to a level not exceeding the pre-rCDI baseline. Collectively, these data support the hypothesis that the restoration of gut microbial communities by FMT for rCDI is associated with an upregulated FXR-FGF pathway, and highlight the potential systemic effect of FMT.

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-C. difficile antibody levels in human sera and in separate preparations of polyclonal IVIg. The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. Herein, a combination of isotype (IgG, IgA IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), precursor form of B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027) and control proteins (Tetanus toxoid and Candida albicans) were determined by protein microarray. Microarrays were probed with sera from individuals with C. difficile infection (CDI), cystic fibrosis (CF) without diarrhea, healthy controls and individuals pre- and post-IVIg therapy for treatment of CDI, combined immunodeficiency disorder and chronic inflammatory demyelinating polyradiculopathy. Significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels were seen between patient groups, commercial preparations of IVIg and sera before and following IVIg administration. A significant correlation was observed between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C. difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, it is anticipated that microarray technology may help optimize and select the most clinicall

Objectives: Despite multiple risk factors and a high rate of colonization for Clostridium difficile, the occurrence of C. difficile infection in patients with cystic fibrosis is rare. The aim of this study was to compare the prevalence of binding C. difficile toxin-specific immunoglobulin (Ig)A, IgG and anti-toxin neutralizing antibodies in the sera of adults with cystic fibrosis, symptomatic C. difficile infection (without cystic fibrosis) and healthy controls. Methods: Subclass-specific IgA and IgG responses to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B only expressing strain (CCUG 20309) and precursor form of B fragment of binary toxin, pCDTb, were determined by protein microarray. Neutralizing antibodies to C. difficile toxins A and B were evaluated using a Caco-2 cell-based neutralization assay. Results: Serum IgA anti-toxin A and B levels and neutralizing antibodies against toxin A were significantly higher in adult cystic fibrosis patients (n=16) compared with healthy controls (n=17) and patients with symptomatic C. difficile infection (n=16); p≤0.05. The same pattern of response prevailed for IgG, except that there was no difference in anti-toxin A IgG levels between the groups. Compared with healthy controls (toxins A and B) and patients with C. difficile infection (toxin A), sera from cystic fibrosis patients exhibited significantly stronger protective anti-toxin neutralizing antibody responses. Conclusion: A superior ability to generate robust humoral immunity to C. difficile toxins in the cystic fibrosis population is likely to confer protection against symptomatic C. difficile infection. This protection may be lost in the post-transplantation setting, where sera-monitoring of anti-C. difficile toxin antibody titers may be of clinical value.

Orbital inflammatory disease is a rare ophthalmic manifestation of Crohn’s disease. Inflammation is characteristically non-specific, involving one or multiple structures of the orbit. Mechanisms of disease and optimal methods of treatment are poorly understood. The aim of this report is to present three cases of orbital involvement in Crohn’s disease. A retrospective case note review of patients with orbital inflammatory disease and Crohn’s disease was performed at our academic centre to determine the clinical, imaging and histopathological features of this condition and its relationship to intestinal Crohn’s disease. Three patients were identified with orbital inflammatory manifestations complicating Crohn’s disease. All patients described were female with active intestinal disease and had a history of treatment with immunosuppressive therapies. Similarities were observed in clinical presentations with variance noted in radiological and histopathological findings. In all cases, symptoms improved with oral corticosteroids or non-steroidal drugs in combination with anti-tumour necrosis factor (anti-TNF) agents. Inflammatory bowel disease-related orbital complications are rare but potentially vision-threatening. It is important to consider mimics of orbital inflammatory disease such as systemic inflammatory disease, malignancy, congenital malformations, infection and trauma when formulating a comprehensive differential diagnosis. Therapeutic intervention is directed towards preservation of vision and orbital function and reducing the acute inflammatory process. Corticosteroids are typically the initial treatment of choice for moderate-to-severe disease, although several classes of immunomodulatory agents have been variably useful in treating this condition. Heightened awareness and close cooperation between gastroenterologists and ophthalmologists is mandatory.

The prevalence of serum antibodies against Clostridium difficile (CD) toxins A and B in healthy populations have prompted interest in evaluating the therapeutic activity of intravenous immunoglobulin (IVIg) in individuals experiencing severe or recurrent C. difficile infection (CDI). Despite some promising case reports, a definitive clinical role for IVIg in CDI remains unclear. Contradictory results may be attributed to a lack of consensus regarding optimal dose, timing of administration and patient selection as well as variability in specific antibody content between commercial preparations. The purpose of this study was to investigate retrospectively the efficacy of three commercial preparations of IVIg for treating severe or recurrent CDI. In subsequent mechanistic studies using protein microarray and toxin neutralization assays, all IVIg preparations were analysed for specific binding and neutralizing antibodies (NAb) to CD antigens in vitro and the presence of anti-toxin NAbs in vivo following IVIg infusion. A therapeutic response to IVIg was observed in 41% (10 of 17) of the CDI patients. Significant variability in multi-isotype specific antibodies to a 7-plex panel of CD antigens and toxin neutralization efficacies were observed between IVIg preparations and also in patient sera before and after IVIg administration. These results extend our current understanding of population immunity to CD and support the inclusion of surface layer proteins and binary toxin antigens in CD vaccines. Future strategies could enhance IVIg treatment response rates by using protein microarray to preselect donor plasma/serum with the highest levels of anti-CD antibodies and/or anti-toxin neutralizing capacities prior to fractionation.

Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotypespecific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated<10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost.

C. difficile infection (CDI) is rarely reported in cystic fibrosis (CF) patients despite frequent hospitalisations and antibiotic usage. Conversely, the prevalence of CDI in inflammatory bowel disease (IBD) has received increased attention. We investigated components of the IgG-specific humoral immune response to C. difficile toxins A and B in patients with C. difficile-associated diarrhoea (CDAD), IBD patients with CDI, CF patients and healthy controls. Serum anti-toxin IgG was determined by ELISA. Circulating antigen-activated B-cells were investigated using Alexa Fluor 488-labelled toxin A and assessed by flow cytometry. Following induction of differentiation of memory B-cells, toxin A- and B-specific antibody secreting cells (ASCs) were quantified using ELISpot. We present the first data showing levels of serum anti-toxin A and B antibodies were significantly higher in patients with CF (without a history of CDI) than in CDAD patients and were stably maintained over time. Notably, the CDAD patients were significantly older than the CF patients. We also show that circulating toxin A-specific memory B-cells (IgD-negative) can be detected in CDAD patients [0.92 (0.09–1.78)%], and were prominent (5.64%, 1.14%) in two CF patients who were asymptomatic carriers of C. difficile. There was correlation between toxin A- and B-specific ASCs, with significantly higher proportions of the latter seen. In some with CDAD, high serum antibody levels were seen to only one of the two toxins. Mucosal secretion of toxin-specific IgG was detected in an additional group of IBD patients with no history of CDI. We conclude that enhanced and stable humoral immune responses to toxins A and B may protect CF and some IBD patients against CDI. The impaired ability to generate strong and/or sustained toxin-specific antibody and memory B-cell responses may increase susceptibility

Clostridium difficile is an anaerobic, Gram-positive, and spore-forming bacterium that is the leading worldwide infective cause of hospital-acquired and antibiotic-associated diarrhea. Several studies have reported associations between humoral immunity and the clinical course of C. difficile infection (CDI). Host humoral immune responses are determined using conventional enzyme-linked immunosorbent assay (ELISA) techniques. Herein, we report the first use of a novel protein microarray assay to determine systemic IgG antibody responses against a panel of highly purified C. difficile-specific antigens, including native toxins A and B (TcdA and TcdB, respectively), recombinant fragments of toxins A and B (TxA4 and TxB4, respectively), ribotypespecific surface layer proteins (SLPs; 001, 002, 027), and control proteins (tetanus toxoid and Candida albicans). Microarrays were probed with sera from a total of 327 individuals with CDI, cystic fibrosis without diarrhea, and healthy controls. For all antigens, precision profiles demonstrated<10% coefficient of variation (CV). Significant correlation was observed between microarray and ELISA in the quantification of antitoxin A and antitoxin B IgG. These results indicate that microarray is a suitable assay for defining humoral immune responses to C. difficile protein antigens and may have potential advantages in throughput, convenience, and cost.

The mechanisms of efficacy for fecal microbiota# transplantation (FMT) in treating recurrent Clostridioides difficile infection (rCDI) remain poorly defined, with restored gut microbiota-bile acid interactions representing one possible explanation. Furthermore, the potential implications for host physiology of these FMT-related changes in gut bile acid metabolism are also not well explored. In this study, we investigated the impact of FMT for rCDI upon signalling through the farnesoid X receptor (FXR)-fibroblast growth factor (FGF) pathway. Herein, we identify that in addition to restoration of gut microbiota and bile acid profiles, FMT for rCDI is accompanied by a significant, sustained increase in circulating levels of FGF19 and reduction in FGF21. These FGF changes were associated with weight gain post-FMT, to a level not exceeding the pre-rCDI baseline. Collectively, these data support the hypothesis that the restoration of gut microbial communities by FMT for rCDI is associated with an upregulated FXR-FGF pathway, and highlight the potential systemic effect of FMT.

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-C. difficile antibody levels in human sera and in separate preparations of polyclonal IVIg. The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. Herein, a combination of isotype (IgG, IgA IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), precursor form of B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027) and control proteins (Tetanus toxoid and Candida albicans) were determined by protein microarray. Microarrays were probed with sera from individuals with C. difficile infection (CDI), cystic fibrosis (CF) without diarrhea, healthy controls and individuals pre- and post-IVIg therapy for treatment of CDI, combined immunodeficiency disorder and chronic inflammatory demyelinating polyradiculopathy. Significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels were seen between patient groups, commercial preparations of IVIg and sera before and following IVIg administration. A significant correlation was observed between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C. difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, it is anticipated that microarray technology may help optimize and select the most clinicall

Orbital inflammatory disease is a rare ophthalmic manifestation of Crohn’s disease. Inflammation is characteristically non-specific, involving one or multiple structures of the orbit. Mechanisms of disease and optimal methods of treatment are poorly understood. The aim of this report is to present three cases of orbital involvement in Crohn’s disease. A retrospective case note review of patients with orbital inflammatory disease and Crohn’s disease was performed at our academic centre to determine the clinical, imaging and histopathological features of this condition and its relationship to intestinal Crohn’s disease. Three patients were identified with orbital inflammatory manifestations complicating Crohn’s disease. All patients described were female with active intestinal disease and had a history of treatment with immunosuppressive therapies. Similarities were observed in clinical presentations with variance noted in radiological and histopathological findings. In all cases, symptoms improved with oral corticosteroids or non-steroidal drugs in combination with anti-tumour necrosis factor (anti-TNF) agents. Inflammatory bowel disease-related orbital complications are rare but potentially vision-threatening. It is important to consider mimics of orbital inflammatory disease such as systemic inflammatory disease, malignancy, congenital malformations, infection and trauma when formulating a comprehensive differential diagnosis. Therapeutic intervention is directed towards preservation of vision and orbital function and reducing the acute inflammatory process. Corticosteroids are typically the initial treatment of choice for moderate-to-severe disease, although several classes of immunomodulatory agents have been variably useful in treating this condition. Heightened awareness and close cooperation between gastroenterologists and ophthalmologists is mandatory.

Objectives: Despite multiple risk factors and a high rate of colonization for Clostridium difficile, the occurrence of C. difficile infection in patients with cystic fibrosis is rare. The aim of this study was to compare the prevalence of binding C. difficile toxin-specific immunoglobulin (Ig)A, IgG and anti-toxin neutralizing antibodies in the sera of adults with cystic fibrosis, symptomatic C. difficile infection (without cystic fibrosis) and healthy controls. Methods: Subclass-specific IgA and IgG responses to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B only expressing strain (CCUG 20309) and precursor form of B fragment of binary toxin, pCDTb, were determined by protein microarray. Neutralizing antibodies to C. difficile toxins A and B were evaluated using a Caco-2 cell-based neutralization assay. Results: Serum IgA anti-toxin A and B levels and neutralizing antibodies against toxin A were significantly higher in adult cystic fibrosis patients (n=16) compared with healthy controls (n=17) and patients with symptomatic C. difficile infection (n=16); p≤0.05. The same pattern of response prevailed for IgG, except that there was no difference in anti-toxin A IgG levels between the groups. Compared with healthy controls (toxins A and B) and patients with C. difficile infection (toxin A), sera from cystic fibrosis patients exhibited significantly stronger protective anti-toxin neutralizing antibody responses. Conclusion: A superior ability to generate robust humoral immunity to C. difficile toxins in the cystic fibrosis population is likely to confer protection against symptomatic C. difficile infection. This protection may be lost in the post-transplantation setting, where sera-monitoring of anti-C. difficile toxin antibody titers may be of clinical value.

The prevalence of serum antibodies against Clostridium difficile (CD) toxins A and B in healthy populations have prompted interest in evaluating the therapeutic activity of intravenous immunoglobulin (IVIg) in individuals experiencing severe or recurrent C. difficile infection (CDI). Despite some promising case reports, a definitive clinical role for IVIg in CDI remains unclear. Contradictory results may be attributed to a lack of consensus regarding optimal dose, timing of administration and patient selection as well as variability in specific antibody content between commercial preparations. The purpose of this study was to investigate retrospectively the efficacy of three commercial preparations of IVIg for treating severe or recurrent CDI. In subsequent mechanistic studies using protein microarray and toxin neutralization assays, all IVIg preparations were analysed for specific binding and neutralizing antibodies (NAb) to CD antigens in vitro and the presence of anti-toxin NAbs in vivo following IVIg infusion. A therapeutic response to IVIg was observed in 41% (10 of 17) of the CDI patients. Significant variability in multi-isotype specific antibodies to a 7-plex panel of CD antigens and toxin neutralization efficacies were observed between IVIg preparations and also in patient sera before and after IVIg administration. These results extend our current understanding of population immunity to CD and support the inclusion of surface layer proteins and binary toxin antigens in CD vaccines. Future strategies could enhance IVIg treatment response rates by using protein microarray to preselect donor plasma/serum with the highest levels of anti-CD antibodies and/or anti-toxin neutralizing capacities prior to fractionation.

The mechanisms of efficacy for fecal microbiota# transplantation (FMT) in treating recurrent Clostridioides difficile infection (rCDI) remain poorly defined, with restored gut microbiota-bile acid interactions representing one possible explanation. Furthermore, the potential implications for host physiology of these FMT-related changes in gut bile acid metabolism are also not well explored. In this study, we investigated the impact of FMT for rCDI upon signalling through the farnesoid X receptor (FXR)-fibroblast growth factor (FGF) pathway. Herein, we identify that in addition to restoration of gut microbiota and bile acid profiles, FMT for rCDI is accompanied by a significant, sustained increase in circulating levels of FGF19 and reduction in FGF21. These FGF changes were associated with weight gain post-FMT, to a level not exceeding the pre-rCDI baseline. Collectively, these data support the hypothesis that the restoration of gut microbial communities by FMT for rCDI is associated with an upregulated FXR-FGF pathway, and highlight the potential systemic effect of FMT.

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-C. difficile antibody levels in human sera and in separate preparations of polyclonal IVIg. The protocol describes the methodological steps involved in sample preparation, printing of arrays, assay procedure and data analysis. In addition, this protocol could be further developed to incorporate diverse clinical samples including plasma and cell culture supernatants. Herein, a combination of isotype (IgG, IgA IgM), subclass (IgG1, IgG2, IgG3, IgG4, IgA1, IgA2), and strain-specific antibodies to highly purified whole C. difficile toxins A and B (toxinotype 0, strain VPI 10463, ribotype 087), toxin B from a C. difficile toxin-B-only expressing strain (CCUG 20309), precursor form of B fragment of binary toxin, pCDTb, ribotype-specific whole surface layer proteins (SLPs; 001, 002, 027) and control proteins (Tetanus toxoid and Candida albicans) were determined by protein microarray. Microarrays were probed with sera from individuals with C. difficile infection (CDI), cystic fibrosis (CF) without diarrhea, healthy controls and individuals pre- and post-IVIg therapy for treatment of CDI, combined immunodeficiency disorder and chronic inflammatory demyelinating polyradiculopathy. Significant differences in toxin neutralization efficacies and multi-isotype specific antibody levels were seen between patient groups, commercial preparations of IVIg and sera before and following IVIg administration. A significant correlation was observed between microarray and enzyme-linked immunosorbent assay (ELISA) for antitoxin IgG levels in serum samples. These results suggest that microarray could become a promising tool for profiling antibody responses to C. difficile antigens in vaccinated or infected humans. With further refinement of antigen panels and a reduction in production costs, it is anticipated that microarray technology may help optimize and select the most clinicall

Orbital inflammatory disease is a rare ophthalmic manifestation of Crohn’s disease. Inflammation is characteristically non-specific, involving one or multiple structures of the orbit. Mechanisms of disease and optimal methods of treatment are poorly understood. The aim of this report is to present three cases of orbital involvement in Crohn’s disease. A retrospective case note review of patients with orbital inflammatory disease and Crohn’s disease was performed at our academic centre to determine the clinical, imaging and histopathological features of this condition and its relationship to intestinal Crohn’s disease. Three patients were identified with orbital inflammatory manifestations complicating Crohn’s disease. All patients described were female with active intestinal disease and had a history of treatment with immunosuppressive therapies. Similarities were observed in clinical presentations with variance noted in radiological and histopathological findings. In all cases, symptoms improved with oral corticosteroids or non-steroidal drugs in combination with anti-tumour necrosis factor (anti-TNF) agents. Inflammatory bowel disease-related orbital complications are rare but potentially vision-threatening. It is important to consider mimics of orbital inflammatory disease such as systemic inflammatory disease, malignancy, congenital malformations, infection and trauma when formulating a comprehensive differential diagnosis. Therapeutic intervention is directed towards preservation of vision and orbital function and reducing the acute inflammatory process. Corticosteroids are typically the initial treatment of choice for moderate-to-severe disease, although several classes of immunomodulatory agents have been variably useful in treating this condition. Heightened awareness and close cooperation between gastroenterologists and ophthalmologists is mandatory.