Antibody targeting of anaerobic bacteria warms cold tumors and improves the abscopal effect of radiotherapy.

BACKGROUND: The combination of immune checkpoint inhibitors with radiotherapy can enhance the immunomodulation by RT and reduce the growth of distant unirradiated tumors (abscopal effect); however, the results are still not very satisfactory. Therefore, new treatment options are needed to enhance this effect. Our previous study showed that the combination of Bifidobacterium (Bi) and its specific monoclonal antibody (mAb) could target and alleviate hypoxia at the tumor site and act as a radiosensitizer. In this study, we explored the anti-tumor efficacy of quadruple therapy (Bi + mAb and RT + αPD-1). The current study also aimed to probe into the complex immune mechanisms underlying this phenomenon. METHODS: Constructed 4T1 breast and CT26 colon cancer tumor models. A comprehensive picture of the impact of constructed quadruple therapy was provided by tumor volume measurements, survival analysis, PET/CT imaging, immune cell infiltration analysis and cytokine expression levels. RESULTS: The abscopal effect was further amplified in the "cold" tumor model and prolonged survival in tumor-bearing mice. Bi can colonized in primary and secondary tumors and direct the mAb to reach the tumor site, activate complement, enhance the ADCC effect and initiate the innate immune response. Then combined with αPD-1 and radiotherapy to stimulate adaptive immune response and synergize with cytokines to expand the immune efficacy and generate effective anti-tumor immune response. CONCLUSIONS: Bi was used as an artificially implanted anaerobic target to cause a transient "infection" at the tumor, causing the tumor to become locally inflamed and "hot", and at the same time, mAb was used to target Bi to enhance the local immune effect of the tumor, and then combined with radiotherapy and αPD-1 to amplify the abscopal effect in multiple dimensions. Therefore, the present study provided a new idea for the multipotent immune-activating function of antibody-targeted anaerobic bacteria for the RT treatment of extensively metastasized cancer patients.

The Surface Microbiome of Clinically Unaffected Skinfolds in Hidradenitis Suppurativa: A Cross-Sectional Culture-Based and 16S rRNA Gene Amplicon Sequencing Study in 60 Patients.

Hidradenitis suppurativa (HS) is a chronic inflammatory disease of the skin associated with specific lesional dysbiotic features. We studied the microbiome of clinically unaffected typical HS sites (armpits, inguinal folds, and gluteal clefts) in 60 patients with HS and 17 healthy controls. A total of 192 samples obtained by swabbing were analyzed by bacterial cultures. Of these, 116 randomly selected samples were studied by 16S rRNA gene amplicon sequencing. Patients and controls showed similar characteristics, except for smoking (87% vs. 6%, respectively). HS skinfolds were characterized by an increased abundance of anaerobes, predominantly Prevotella, but also Actinomyces, Campylobacter ureolyticus, and Mobiluncus, contrasting with a lower abundance of skin commensals such as Staphylococcus epidermidis, a major component of the skin microbiome; Kocuria; and Micrococcus luteus. The following three independent factors were associated with an abundance of high anaerobes by multivariate analysis: samples originating from patients with HS patients (P = 2.1 × 10-4); body mass index (P = 5 × 10-5); and the sampling site, the gluteal cleft being the most anaerobic area, followed by inguinal folds and axilla (P = 3 × 10-6). The microbiome of clinically unaffected HS skinfolds is reminiscent, albeit to a minor extent, of the microbiome of chronic suppurative HS lesions and may fuel inflammation at a preclinical stage of the disease.

Evaluation of serological markers to differentiate between ulcerative colitis and Crohn's disease: pANCA, ASCA and agglutinating antibodies to anaerobic coccoid rods.

BACKGROUND: Accurate diagnosis of inflammatory bowel disease, in particular the differentiation between ulcerative colitis and Crohn's disease, is important for treatment and prognosis. Several serological markers have been used as non-invasive diagnostic tools in inflammatory bowel disease patients both to differentiate ulcerative colitis from Crohn's disease and to define patient subgroups. AIM: To evaluate the diagnostic accuracy of three serological tests in differentiating ulcerative colitis from Crohn's disease by single or combined use. METHODS: Sera from 51 patients with clinically well-defined ulcerative colitis and 50 patients with clinically well-defined Crohn's disease were analysed. Detection assays for the presence of perinuclear anti-neutrophil cytoplasmatic antibodies (pANCA), antibodies against (ASCA) and serum agglutinating antibodies to anaerobic coccoid rods were studied. Sensitivity, specificity, predictive values and likelihood ratios of each of these serological tests were determined. RESULTS: In supporting the diagnosis of ulcerative colitis, the sensitivity and specificity of the pANCA test were 63% and 86%, respectively. The ASCA test (immunoglobulin A or immunoglobulin G positive) for diagnosing Crohn's disease had a sensitivity of 72% and a specificity of 82%. The sensitivity of antibodies to anaerobic coccoid rods in diagnosing Crohn's disease was 52%, whereas specificity was 90%. A combination of pANCA-positive and ASCA-negative results in the case of ulcerative colitis showed a sensitivity and specificity of 51% and 94%, respectively. However, for ASCA-positive and pANCA-negative results in the case of Crohn's disease, sensitivity was 64% and specificity was 94%. The combination of all three tests increased positive predictive value and specificity to 100% for both ulcerative colitis and Crohn's disease. In Crohn's disease patients, positive pANCA was correlated with colonic involvement. No correlation was found between the presence of any of these antibodies and disease activity, duration and behaviour or medical treatment. CONCLUSIONS: The value of these serological tests in differentiating ulcerative colitis from Crohn's disease is limited when used separately but, by combining two or more tests, the positive predictive value and specificity can be improved substantially. These tests might be of help in studying disease heterogeneity, and may contribute to defining various subgroups of patients with different pathogeneses.

Matrilysin (matrix metalloproteinase-7) expression in human junctional epithelium.

Matrilysin is a matrix metalloproteinase expressed in exocrine and mucosal epithelium in many human tissues. Immunohistochemical staining showed that matrilysin is expressed in suprabasal cells of junctional epithelium facing the teeth and in epithelial cell rests of Malassez. No matrilysin expression was seen in the periodontal pocket tissue. In a tissue culture model mimicking junctional epithelium, matrilysin expression was also observed in suprabasal epithelial cells. Of 13 anaerobic oral bacterial species tested, F. nucleatum, F. necrophorum, P. endodontalis, and P. denticola stimulated matrilysin expression in porcine periodontal ligament epithelial cells from 2.5- to 5.7-fold, compared with untreated cells. The enzyme was localized in intracytoplasmic vesicles that also reacted with antibodies against lysosomal membrane protein h-lamp-1. The results indicate that matrilysin may play an important role in the normal physiology of junctional epithelium.

Immune response in lymphocyte cultures stimulated by oral bacteria preparations.

Lymphocyte cultures were used as an in vitro experimental model to get a deeper insight into immune response to oral bacteria in periapical granulomas. Lymphocytes isolated from leucocyte concentrate were in lymphocyte cultures stimulated by antigen preparations of oral bacteria. Lymphocyte subsets that have developed in lymphocyte cultures after a week of stimulation were analysed by flow cytometry. A significant increase in expression of INF-gamma molecules in CD3+ cells stimulated by antigen preparations of oral streptococci was found, compared with negative control. On the other hand we observed a significant increase in expression of IL-4 in CD3+ cells stimulated by antigens of anaerobic bacteria, compared with negative control. Our results show that antigens of oral streptococci in in vitro lymphocyte cultures induce the differentiation of T helper cells into Th2 cells and that antigen preparations of anaerobic bacteria induce the differentiation of T helper cells into Th1 cells. Furthermore, an increased expression of HLA-DR molecules on CD8+ T cells stimulated by antigens of oral streptococci was found, compared with negative control.

Determination de una técnica de referencia para la susceptibilidad antimicrobiana de bacterias anaeróbicas de importancia clínica / Determination antimicrobial susceptibility test of anaerobes in reference centres

La selección de antibióticos para el tratamiento de infecciones por microorganismos anaeróbicos se hace en forma empírica en la mayoría de los casos; por tanto; el empleo de técnicas para determinar la susceptibilidad antimicrobiana es controvertido y sólo se realiza en centros de referencia. Con la aparición de cepas resistentes a los antimicrobianos de elección, es necesario determinar entre las técnicas estandarizadas una que pueda ser realizada en un laboratorio con cierta infraestructura para el trabajo en bacteriología clínica. Se realizaron cuatro pruebas estandarizadas por el NCCLS: elución de disco en caldo, macrodilución, microdilución y dilución en agar, empleando cuatro cepas de referencia. Los datos obtenidos fueron analizados para determinar la reproducibilidad, la concordancia y la complejidad de las pruebas. Empleando los criterios analizados, se obtuvo en orden descendente: en reproducibilidad, la dilución en agar, microdilución, macrodilución, y elución; en concordancia, la dilución en agar, macrodilución, elución y microdilución; y en complejidad, dilución en agar, microdilución, macrodilución y elución. Debido a la variedad de los resultados obtenidos no se pudo recomendar una sola técnica para la determinación de la susceptibilidad de microorganismos anaeróbicos de importancia clínica; en algunos casos y teniendo en cuenta el microorganismo y el antibiótico a probar, la realización de por lo menos dos de ellas permitiría esa determinación

Biology of acute and chronic graft-versus-host reactions: predictive value of studies in experimental animals.

Experimental research on graft-versus-host disease (GVHD) with laboratory animals has been performed mainly with rodents, rhesus monkeys and dogs. The basic immunological mechanisms operative in GVHD are largely similar in these three species and in human patients, although the patterns of GVHD in the three animal species show differences. The predictive value for clinical GVHD of the results obtained in the different animals species is analysed for the three main variables: namely, histocompatibility, T cell numbers in the graft and the intestinal microflora. Rhesus monkeys score highest as regards clinical relevance for the first two variables. With regards to the unravelling of detailed mechanisms of the influence of the microflora, none of the three animal species is likely to provide the information needed for identification of the bacterial species involved in the induction of GVHD in human patients.

Adequate function of the immune system and physiological microflora are closely correlated.

It is already known that physiological microflora of the digestive system plays an important role in local immunity. Studies on immunomodulating activity of some autoantibodies have shown that drugs affecting intestinal microflora possess potent immunosuppressive effect on systemic immunity. These observations inspired to recent investigation on the mechanisms of the influence of digestive tract bacteria on the immune system.

Serum antibodies to peptidoglycan-polysaccharide complexes from the anaerobic intestinal flora in patients with Crohn's disease.

Soluble peptidoglycan-polysaccharide complexes (PPC) isolated from feces of a healthy subject shared antigens with 22 out of 40 anaerobic intestinal bacteria and, therefore, these could be considered as flora derived. IgG, IgA and IgM antibodies against PPC were found in sera from healthy subjects, patients with Crohn's disease (CD) and other patients. In sera of CD patients with agglutinating antibodies to 4 anaerobic intestinal bacteria (used as an aid for diagnosis of CD) IgG and IgA levels to PPC were higher than in CD patients without these antibodies.

Susceptibilidad a los antibióticos de los principales grupos de bacterias aisladas de pacientes con sepsis intraabdominal en dos hospitales generales de Costa Rica / Antibiotic susceptibility of the principal groups of bacteria isolated from patients with intra-abdominal sepsis in two Costa Rican general hospitals

Durante 1984 y 1985, se recolectó muestras de 68 pacientes con sepsis intra-abdominal, para estudiar la presencia de bacterias anaerobias, facultativas y determinar la sensibilidad a los antibióticos de los principales organismos encontrados. Bacteroides fragilis y Escherichia coli fueron las bacterias aisladas con mayor frecuencia. Todas las cepas de B. Fragilis analizadas, fueron sensibles a tinidazol, metronidazol y al cloranfenicol; sin embargo se encontro porcentajes de resistencia del 97,2 por ciento a la penicilina G, 8,3 por ciento a la clindamicina y 2,7 por ciento a la doxiciclina. Entre las cepas de E. coli estudiadas, los porcentajes de resistencia fueron de 55,9 por ciento al trimetoprin-sulfametoxazol. Los resultados obtenidos hacen ver la importancia de mantener un programa de vigilancia de la sensibilidad a los agentes bacterianos, tanto para las bacterias facultativas como anaerobias

Induction of chronic arthritis in rats by cell wall fragments of anaerobic coccoid rods isolated from the faecal flora of patients with Crohn's disease.

Crohn's disease and ulcerative colitis are accompanied by seronegative arthritis in about one fifth of the cases. In the present study, cell wall fragments from major residents such as Eubacterium, Coprococcus and Peptostreptococcus species, isolated from the faecal flora of patients with Crohn's disease, were tested for properties to induce chronic arthritis in Lewis rats. Cell wall fragments from Eubacterium contortum strains Me44 and Me47 were found to induce chronic arthritis; Peptostreptococcus productus strain C 18 cell wall fragments induced acute self-limiting arthritis. Coprococcus comes strain Me46 cell walls, on the other hand, were found to be lethal to the majority of rats inoculated, whereas those which survived did not develop acute or chronic arthritis. The results indicate that intraperitoneal injection of a single dose of cell wall fragments from bacteria that are major residents of the human anaerobic faecal flora can induce chronic inflammatory joint disease in the rat.

Antibodies to Coprococcus comes in sera of patients with Crohn's disease. Isolation and purification of the agglutinating antigen tested with an ELISA technique.

Previous studies showed that agglutinating antibodies to Coprococcus comes, an anaerobic Gram-positive coccoid rod isolated from the faecal flora of patients with Crohn's disease, are more frequently found in sera of Crohn patients than in ulcerative colitis patients and healthy subjects. Isolation of the antigen may be useful in developing a more sensitive and specific diagnostic test. The present study describes first a method to improve the presentation of the relevant agglutinating antigen by the bacterium and second, the purification by column chromatography of a relatively crude antigen extract of C. comes described previously by Hazenberg et al. (1). Comparative results with the agglutination reactions and ELISA technique of extensive series of patients with Crohn's disease and healthy subjects have shown that the agglutinating antigen of C. comes has been isolated. Although the present ELISA technique cannot replace the simple and reliable agglutination reaction for screening purposes, the purified antigen will allow further immunological studies and it is to be hoped that a deeper insight into pathogenesis of the disease will be gained.

Antibody response to anaerobic coccoid rods in Crohn's disease.

The IgG and IgM specific antibodies against a panel of 23 anaerobic gut bacteria were examined in Crohn's disease, ulcerative colitis, and healthy controls. Four of the organisms, Bifidobacterium bifidum, Coprococcus comes (ME46), Coprococcus comes (Sp4), and Eubacterium limosum gave abnormal antibody titres in Crohn's disease compared with those of controls. In Crohn's disease specific IgG antibodies to three of the organisms were low and the IgM antibodies were higher than those of controls. IgM antibodies were also raised in ulcerative colitis. Antigenic cross reactivity could be shown between some of these organisms. The possible clinical importance of these abnormal antibody responses to specific organisms is unexplained.

The human faecal flora and Crohn's disease.

The aetiology of Crohn's disease (CD), a chronic inflammatory disease of the intestines, is not known but a genetic predisposition to CD has been well demonstrated. Studies on the isolation of bacteria from tissues and the faecal flora from CD patients are reviewed and their results compared with those from our laboratory. Serum antibodies against a variety of bacteria have been described but none has been found to be specific for CD. In our laboratory, higher numbers of coccoid anaerobes have been found in the faecal flora of CD patients. The use of agglutination reactions with four coccoid anaerobes as a diagnostic aid for CD is reviewed. No evidence has been found, however, for a primary role of the agglutinating antibodies in the pathogenesis of CD. On the other hand, one of the coccoid strains has been found to have several characteristics suggesting a role in CD, such as activation of the alternative pathway of complement, the failure of opsonization by specific IgG antibody and the binding of (non-specific) IgG onto the bacteria by the Fc portion. The composition of the faecal flora has been found to depend on genetic characteristics of the host. It is suggested that the genetic predisposition to CD is based on the genetically determined indigenous faecal flora present.

Immune responses to anaerobic gram-negative bacteria in health and disease.

In the serum of healthy and sick persons antibodies to Bacteroidaceae and Veillonella have been detected. The antibody titres to these anaerobic bacteria are commonly low, in adults, however, higher than in children. High antibody titres could be found in patients suffering from Crohn's disease and in cancer patients. Immune responses to anaerobic bacteria occur in periodontal processes, too. These antibodies belong mainly to the IgM class of immunoglobulins. On the contrary, antibodies observed in patients with sepsis belong to the IgG class.