Objective Visual Analog Scale for Biopsy Diagnosis of Helicobacter pylori Infection in Clinical Practice.

Historic and current pathology society guidelines recommend using visual gestalt to identify substantial inflammatory cell infiltrate in Helicobacter pylori gastritis, but these scales were subjectively designed. This study aims to objectively investigate the density of inflammation that justifies additional workup for H. pylori infection. We retrospectively identified 2 patient cohorts who had undergone endoscopy with gastric biopsies; 1 with H. pylori infection (n=66), confirmed with a positive stool antigen test and/or Campylobacter-like organism test, and 1 without infection (n=81). Antral and body biopsies were selected from each case, if available, and stained with MUM-1 to highlight mucosal plasma cells. Digital analysis was performed to calculate the number of plasma cells/mm2, termed the "inflammatory score" (IS). Patients with H. pylori infection had an average of 1289 plasma cells/mm2 in the antrum and 835 plasma cells/mm2 in the body, compared with 346 plasma cells/mm2 in the antrum and 178 plasma cells/mm2 in the body in patients without infection. IS cut-off values for a positive infection were 714 plasma cells/mm2 in the antrum and 316 plasma cells/mm2 in the body, with high sensitivities and specificities in both the antrum (92%, 92%) and body (85%, 84%), respectively. A visual analog scale was created to provide a histologic correlate of the observed IS ranges and cut-offs. This practical and objective scale is associated with a high sensitivity and specificity for diagnosing H. pylori infection and justifies moving away from upfront universal H. pylori testing in routine clinical practice.

Syphilis of the Aerodigestive Tract.

Syphilis, a sexually transmitted infection caused by the Gram-negative bacterium Treponema pallidum, is increasing in prevalence in the United States. It has been our experience that primary and secondary syphilis of the aerodigestive tract can afflict a large age spectrum with varied clinical and histopathologic findings, which can lead to diagnostic problems and frequent misdiagnosis. In this study, we describe the histopathologic patterns of syphilis of the aerodigestive tract to expand awareness of its varied appearance. We identify 3 patterns of inflammatory response to syphilis: plasma cell-rich, lymphohistiocytic, and lymphoma-like. We also report the presence of immunoglobulin G4-predominant plasma cells in the inflammatory response as a potential mimicker of immunoglobulin G4-related disease. Lastly, we found that use of T. pallidum immunohistochemical stain is more reliable than Steiner silver stain at the identification of spirochetes. Our study highlights that despite convention, plasma cells are not always abundant in syphilis. Awareness of the histopathologic range of syphilis in the aerodigestive tract by the surgical pathologist can lead to the correct diagnosis and guide appropriate treatment.

Immunogenesis in White Mice Infected with Yersinia pestis with Different Plasmid Composition.

Morphological changes in the immunocompetent organs of white mice with experimental plague infection manifested in activation of the immune response of different degree and pathological process of different severity that depended on the plasmid composition of Y. pestis. Widening of the T-dependent zones in the immune organs of white mice infected with isogenic strains of Y. pestis with different plasmid composition attests to activation of cellular immunity. Our findings allow considering Y. pestis subsp. altaica I-2948/3, Y. pestis subsp. pestis I-3479 and Y. pestis subsp. pestis I-3480 as promising candidates for vaccine strains.

Increased plasmacytic differentiation in gastric mucosa-associated lymphoid tissue lymphomas: Helicobacter pylori eradication response and IgG4+ plasma cell association.

Gastric mucosa-associated lymphoid tissue (MALT) lymphoma is a rare extranodal marginal zone B-cell lymphoma that is often associated with plasmacytic differentiation. However, the clinicopathological characteristics of gastric MALT lymphoma with increased plasmacytic differentiation have not yet been studied. To assess the clinicopathological implications of gastric MALT lymphoma with increased plasmacytic differentiation, 36 cases with increased plasmacytic differentiation and a control group of 16 cases with minimal plasmacytic differentiation were retrospectively collected from 65 primary gastric MALT lymphomas (2010-2012). The hematoxylin and eosin slides were reviewed, and IgG, IgG4, and κ and λ immunohistochemical staining was performed. Clinicopathological differences between the 2 groups were compared using the χ2 test and odds ratios. Logistic regression analyses were used to evaluate resistance to Helicobacter pylori eradication therapy. Increased plasmacytic differentiation is significantly correlated with the H pylori eradication response (94.4% versus 66.7%, P=.018), lower frequency of relapse (5.6% versus 35.7%, P=.014), the presence of more than one IgG4+ cell per high-power field (27.8% versus 0%, P=.022), and light-chain restriction (33.3% versus 6.2%, P=.044). Univariable logistic regression indicated that negative H pylori status (P=.016) and minimal plasmacytic differentiation (P=.019) were statistically significant predictive factors for resistance to H pylori eradication. Multivariable logistic regression analyses identified no statistically significant predictive factors. However, H pylori negativity and minimal plasmacytic differentiation showed a statistical trend toward significance (P=.078 and P=.09). Gastric MALT lymphomas with increased plasmacytic differentiation have different clinicopathological characteristics, and plasmacytic differentiation is associated with H pylori eradication response.

Eosinophils may play regionally disparate roles in influencing IgA(+) plasma cell numbers during large and small intestinal inflammation.

BACKGROUND: Eosinophils are innate immune cells present in the intestine during steady state conditions. An intestinal eosinophilia is a hallmark of many infections and an accumulation of eosinophils is also observed in the intestine during inflammatory disorders. Classically the function of eosinophils has been associated with tissue destruction, due to the release of cytotoxic granule contents. However, recent evidence has demonstrated that the eosinophil plays a more diverse role in the immune system than previously acknowledged, including shaping adaptive immune responses and providing plasma cell survival factors during the steady state. Importantly, it is known that there are regional differences in the underlying immunology of the small and large intestine, but whether there are differences in context of the intestinal eosinophil in the steady state or inflammation is not known. RESULTS: Our data demonstrates that there are fewer IgA(+) plasma cells in the small intestine of eosinophil-deficient ΔdblGATA-1 mice compared to eosinophil-sufficient wild-type mice, with the difference becoming significant post-infection with Toxoplasma gondii. Remarkably, and in complete contrast, the absence of eosinophils in the inflamed large intestine does not impact on IgA(+) cell numbers during steady state, and is associated with a significant increase in IgA(+) cells post-infection with Trichuris muris compared to wild-type mice. Thus, the intestinal eosinophil appears to be less important in sustaining the IgA(+) cell pool in the large intestine compared to the small intestine, and in fact, our data suggests eosinophils play an inhibitory role. The dichotomy in the influence of the eosinophil over small and large intestinal IgA(+) cells did not depend on differences in plasma cell growth factors, recruitment potential or proliferation within the different regions of the gastrointestinal tract (GIT). CONCLUSIONS: We demonstrate for the first time that there are regional differences in the requirement of eosinophils for maintaining IgA+ cells between the large and small intestine, which are more pronounced during inflammation. This is an important step towards further delineation of the enigmatic functions of gut-resident eosinophils.

BH3-only protein Bim is associated with the degree of Helicobacter pylori-induced gastritis and is localized to the mitochondria of inflammatory cells in the gastric mucosa.

BH3-only protein, Bim, is a pro-apoptotic protein that mediates mitochondria-dependent cell death. However, the role of Bim in Helicobacter pylori-associated gastritis remains unclear. This study aimed to assess the cellular localization of Bim and its possible role in H. pylori-induced gastritis. The study was conducted on biopsy specimens obtained from 80 patients who underwent upper gastrointestinal endoscopy (H. pylori-negative: n=30, positive: n=50). Association between Bim mRNA expression and severity of gastritis was evaluated and the localization of Bim was examined by immunofluorescence. Bim mRNA expression was positively correlated with the degree of gastritis, as defined by the Sydney system. Immunohistochemical analysis confirmed increased Bim expression in H. pylori-infected gastric mucosa compared with uninfected mucosa in both humans and mice. Bim localized in myeloperoxidase- and CD138-positive cells of H. pylori-infected lamina propria and submucosa of the gastric tract, indicating that this protein is predominantly expressed in neutrophils and plasma cells. In contrast, Bim did not localize in CD20-, CD3-, or CD68-positive cells. Bim was expressed in the mitochondria, where it was partially co-localized with activated Bax and cleaved-PARP. In conclusion, Bim is expressed in neutrophils and plasma cells in H. pylori-associated gastritis, where it may participate in the termination of inflammatory response by causing mitochondria-mediated apoptosis in specific leucocytes.

Paediatric nodal marginal zone B-cell lymphadenopathy of the neck: a Haemophilus influenzae-driven immune disorder?

Many hyperplasias and lymphomas of marginal zone B-cells are associated with infection. We identified six children and one adolescent with cervical lymphadenopathy showing prominent polyclonal nodal marginal zone hyperplasia (pNMZH) and four adolescents with monoclonal paediatric nodal marginal zone lymphoma (pNMZL). The clonality status was assessed using BIOMED-2-IG PCR analysis. Haemophilus influenzae was identified in all six cases of pNMZH that could be tested by direct culture (N = 3) or a very sensitive PCR for the H. influenzae gyrase gene in frozen materials (N = 5). H. influenzae was not detected in three pNMZLs and 28 non-specific reactive cervical lymph nodes of age-matched controls, except for a single control node that was obtained during oropharyngeal surgery for a cleft palate showing very low copy numbers of H. influenzae. pNMZH patients were younger than pNMZL patients (median age 12 versus 21 years). pNMZH showed a prominent nodular appearance with variable fibrosis without acute inflammation. Within the nodules, the expanded germinal centres and variably sized marginal zones were colonized by activated B-cells with weak expression of IgD and lack of CD10 and/or BCL6 expression. Some areas showed skewed light chain expression in plasma cells (4/5 cases lambda). In four cases tested, this was confirmed by flow cytometry for surface Ig (3/4 cases lambda). In contrast, pNMZL showed more extensive expansion of marginal zones by centrocytoid cells and often expression of BCL2 protein. Several H. influenzae strains are known to interact with the constant part of IgD on human B-cells, leading to their polyclonal proliferation and activation. We speculate that in vivo stimulation of IgD+ marginal zone B-cells by this bacterium may be implicated in this particular lymphadenopathy that should be distinguished from monoclonal pNMZL.

Impact of dietary deviation on disease progression and gut microbiome composition in lupus-prone SNF1 mice.

Environmental factors, including microbes and diet, play a key role in initiating autoimmunity in genetically predisposed individuals. However, the influence of gut microflora in the initiation and progression of systemic lupus erythematosus (SLE) is not well understood. In this study, we have examined the impact of drinking water pH on immune response, disease incidence and gut microbiome in a spontaneous mouse model of SLE. Our results show that (SWR × NZB) F1 (SNF1 ) mice that were given acidic pH water (AW) developed nephritis at a slower pace compared to those on neutral pH water (NW). Immunological analyses revealed that the NW-recipient mice carry relatively higher levels of circulating autoantibodies against nuclear antigen (nAg) as well as plasma cells. Importantly, 16S rRNA gene-targeted sequencing revealed that the composition of gut microbiome is significantly different between NW and AW groups of mice. In addition, analysis of cytokine and transcription factor expression revealed that immune response in the gut mucosa of NW recipient mice is dominated by T helper type 17 (Th17) and Th9-associated factors. Segmented filamentous bacteria (SFB) promote a Th17 response and autoimmunity in mouse models of arthritis and multiple sclerosis. Interestingly, however, not only was SFB colonization unaffected by the pH of drinking water, but also SFB failed to cause a profound increase in Th17 response and had no significant effect on lupus incidence. Overall, these observations show that simple dietary deviations such as the pH of drinking water can influence lupus incidence and affect the composition of gut microbiome.

Acrodermatitis chronica atrophicans with pseudolymphomatous infiltrates.

In this study, we describe the clinicopathologic features of pseudolymphomatous infiltrates found within lesions of acrodermatitis chronica atrophicans (ACA). We studied 11 patients (10 females, 1 male, age range 60-88 years). The diagnosis of ACA in all cases was confirmed by clinicopathologic correlation and positive serology for Borrelia. Histopathologic examination revealed prominent, pseudolymphomatous inflammatory cell infiltrates in all cases, with 2 distinct patterns. Eight of 11 cases showed a band-like lymphocytic infiltrate, exocytosis of lymphocytes and a fibrotic papillary dermis, similar to features seen in mycosis fungoides. The other 3 cases showed dense, nodular-diffuse dermal infiltrates with many plasma cells and without germinal centers. The plasma cells expressed both kappa and lambda immunoglobulin light chains with a polyclonal pattern in all 3 cases. In conclusion, ACA may present with pseudolymphomatous infiltrates showing both a T-cell and, less frequently, a B-cell pattern. These lesions need to be distinguished from a cutaneous lymphoma. In the context of the knowledge of Borrelia-associated cutaneous lymphomas, follow-up seems advisable in these cases.

Solitary plasmacytoma of the tonsillar site associated with actinomyces infection: the possible role of IL-6.

ExtraMedullary Plasmacytoma (EMP) is a rare plasma cell tumor. It can occur in the upper aerodigestive tract and presents as a large nodule causing local compressive symptoms. A 79-year old woman presented to Otorhinolaryngology Department with progressive hearing loss and no other symptoms. Following PET/TC examination due to the suspicion of a lymphoproliferative disease, the patient underwent tonsillectomy and the diagnosis of solitary EMP was formulated. In addition to that, the histological examination of the tonsillar tissue revealed large colonies of filamentous bacteria, showing abundant sulphur granules and Splendore-Hoeppli phenomenon; these evidences indicating the presence of a chronic Actinomyces infection. Immunohistochemical analysis demonstrated a marked IL-6 immunoreactivity of the neoplastic plasma cells. Interestingly, a marked IL-6 immunoreactivity was also found in the tissue surrounding the Actinomyces colonies. In the present study we report for the first time a solitary EMP associated with Actinomycosis. It is tempting to speculate that the unsuspected and untreated Actinomyces infection, through chronic IL-6 production, could contribute to the neoplastic transformation of plasma cells.

Russell body duodenitis: a histopathological and molecular approach to a rare clinical entity.

Russell bodies are pink eosinophilic accumulations within plasma cells. To date, two hypotheses have attempted to elucidate the biological events behind the formation of these bodies. One theory sustains that such bodies constitute cytoplasmic accumulation of immunoglobulin derivatives contained in the perinuclear cistern of the smooth endoplasmic reticulum because of an increased synthesis or altered secretion. On the other hand, since its initial description in the medical literature, several authors have attributed the formation of such bodies to the presence of microorganisms such as in the case of Russell body gastritis and its association to Helicobacter pylori infection. In an attempt to possibly characterize the presence of an infectious organism, we performed a thorough biomolecular analysis on a case of a 69-year-old female presenting with Russell body duodenitis which, to the best of our knowledge, constitutes the second report of this clinical entity in the English literature. In light that the events behind formation of such bodies in H. pylori-negative individuals remain unclear, we hypothesize on the possible pathways that could have led to their reactive mechanical and immune derivation.

Pathogen-specific circulating plasmablasts in patients with pneumonia.

Lower respiratory tract infections (LRTI) are the leading cause of death world-wide, with Streptococcus pneumoniae (Pnc) as the most prevalent pathogen. Local immune mechanisms appear central to protection against the disease, yet they are poorly characterized. Infections at other, non-respiratory mucosal sites are associated with a transient circulation of mucosa-originating lymphocytes from the mucosal site to blood and back to the mucosa. The present study explored whether pathogen-specific plasmablasts appear in the circulation also in patients with infection of the lower respiratory tract. 16 patients with bacteremic Pnc pneumonia and 14 healthy volunteers were explored for circulating plasmablasts secreting antibodies against their own pathogenic Pnc strain isolated in blood cultures (patients) or against several pathogenic strains from pneumonia patients (14 controls) or a mixture of nine different purified pneumococcal polysaccharides (8 controls). Both patients and volunteers were studied for all plasmablasts. The cells were identified with ELISPOT as Pnc-specific antibody-secreting cells (ASC) and as all immunoglobulin-secreting cells (ISC). High numbers of circulating Pnc-specific ASC were found in the acute phase of the disease in all patients with pneumonia (median 97 ASC/10(6) PBMC), but in none of the controls. IgG isotype predominated in 9/16 patients. The numbers of ISC were significantly higher in the patients than in the healthy controls, yet Pnc-specific ASC only accounted for 0.7% of all the patients' ISC.The present study is the first to show that antigen-specific plasmablasts appear in the circulation in pneumonia, suggesting that pulmonary lypmhocytes recirculate in humans. Assessing these cells provides a novel tool for studying immune response to antigens encountered at the LRT.

Specific in situ visualization of plasma cells producing antibodies against Porphyromonas gingivalis in gingival radicular cyst: application of the enzyme-labeled antigen method.

The enzyme-labeled antigen method was applied to visualize plasma cells producing antibodies to Porphyromonas gingivalis, flora of the human oral cavity. Antibodies to P. gingivalis have reportedly been detected in sera of patients with periodontitis. Biotinylated bacterial antigens, Ag53, and four gingipain domains (Arg-pro, Arg-hgp, Lys-pro, and Lys-hgp) were prepared by the cell-free protein synthesis system using the wheat germ extract. In paraformaldehyde-fixed frozen sections of rat lymph nodes experimentally immunized with Ag53-positive and Ag53-negative P. gingivalis, plasma cells were labeled with biotinylated Arg-hgp and Lys-hgp. Antibodies to Ag53 were detected only in the nodes immunized with Ag53-positive bacteria. In two of eight lesions of gingival radicular cyst with inflammatory infiltration, CD138-positive plasma cells in frozen sections were signalized for Arg-hgp and Lys-hgp. An absorption study using unlabeled antigens confirmed the specificity of staining. The AlphaScreen method identified the same-type antibodies in tissue extracts but not in sera. Antibodies to Ag53, Arg-pro, and Lys-pro were undetectable. In two cases, serum antibodies to Arg-hgp and Lys-hgp were AlphaScreen positive, whereas plasma cells were scarcely observed within the lesions. These findings indicate the validity of the enzyme-labeled antigen method. This is the very first application of this novel histochemical technique to human clinical samples.

IgM production by bone marrow plasmablasts contributes to long-term protection against intracellular bacterial infection.

IgM responses are well known to occur early postinfection and tend to be short-lived, which has suggested that this Ig does not significantly contribute to long-term immunity. In this study, we demonstrate that chronic infection with the intracellular bacterium Ehrlichia muris elicits a protective, long-term IgM response. Moreover, we identified a population of CD138(high)IgM(high) B cells responsible for Ag-specific IgM production in the bone marrow. The IgM-secreting cells, which exhibited characteristics of both plasmablasts and plasma cells, contributed to protection against fatal ehrlichial challenge. Mice deficient in activation-induced cytidine deaminase, which produce only IgM, were protected against fatal ehrlichial challenge infection. The IgM-secreting cells that we have identified were maintained in the bone marrow in the absence of chronic infection, as antibiotic-treated mice remained protected against challenge infection. Our studies identify a cell population that is responsible for the IgM production in the bone marrow, and they highlight a novel role for IgM in the maintenance of long-term immunity during intracellular bacterial infection.

Decreased IgA antibody production in the stomach of gastric adenocarcinoma patients.

Gastric adenocarcinoma is closely associated with Helicobacter pylori infection. It is also much more frequent in patients with common variable immunodeficiency or selective IgA-deficiency than in the general population. To investigate a possible link between local antibody production and gastric tumors, we studied gastric B cell infiltration and local IgA production in patients with H. pylori induced gastric adenocarcinomas. These studies showed that total and H. pylori-specific IgA antibody levels were substantially lower in gastric tissue from the cancer patients compared to those from asymptomatic H. pylori carriers. However, serum IgA levels were similar in the cancer patients and asymptomatic carriers. As could be expected, H. pylori infected asymptomatic carriers had considerably increased IgA antibody levels compared to uninfected subjects. We conclude that patients suffering from gastric adenocarcinoma have a dramatically decreased local IgA production in the stomach compared to asymptomatic H. pylori infected individuals.

Possible relationship between intestinal barrier function and formation of pigment gallstones in hamsters.

BACKGROUND: The presence of bacteria in bile is an important factor in the formation of pigment gallstones. The bile of healthy people is sterile and bacteria in the biliary system come from endogenous infection from the gut. Yet, the route of bacterial translocation into the bile duct is still unclear. Theoretically, two routes exist: one is through the intestinal barrier and the other is by direct reflux from the sphincter of Oddi. This study was undertaken to explore the relationship between the effectiveness of intestinal barrier and the formation of pigment gallstones in hamsters. METHODS: Thirty-two hamsters were divided into an experimental and a control group, with 16 hamsters in each group. A low protein and high cellulose diet was given for 6 weeks to induce the formation of pigment gallstones in the experimental group (PS) and a normal diet was given to the control group (CON). Morphological changes, changes in the levels of serum endotoxin and diamine oxidase, and changes in the numbers of B lymphocytes, plasma cells and secretory immunoglobin A (sIgA) in the intestinal mucosa were assessed after 6 weeks. RESULTS: Four hamsters died during lithogenesis and body weight decreased in the PS group. Pigment gallstones were found in 11 hamsters at the end of the experiment, giving a lithogenesis rate of 91.67%. The serum endotoxin level before and after gallstone formation in the PS group was 0.2960+/-0.1734 U/ml and 8.2964+/-4.6268 U/ml, respectively (P<0.05). The blood diamine oxidase level before and after gallstone formation in the PS group was 2.6333+/-0.8037 U/ml and 3.3642+/-0.9545 U/ml, respectively (P<0.05). The numbers of B lymphocytes, plasma cells and sIgA in the intestinal mucosa in the PS group were 71.56+/-2.89, 68.65+/-2.09 and 27.56+/-1.07, respectively, and were significantly decreased compared with the corresponding values in the CON group (94.25+/-3.69, 93.47+/-3.98 and 42.57+/-1.96, respectively, P<0.05). CONCLUSIONS: A low protein and high cellulose diet can markedly reduce intestinal barrier function and facilitate the formation of pigment gallstones. The decrease of intestinal barrier function may take part in the formation of pigment gallstones.

Immunopathological responses and kinetics of Brucella melitensis Rev 1 infection after subcutaneous or conjunctival vaccination in rams.

The innocuousness of the Brucella melitensis Rev 1 live attenuated vaccine strain has never been fully assessed in rams. The immunopathological responses and the kinetics and distribution of the infection induced by this strain were determined after subcutaneous or conjunctival vaccination in both young (3-4 months old) and adult (12 months old) rams. At regular intervals after vaccination the animals were bled for serological studies, and slaughtered for both pathological and bacteriological examinations. The serological response after conjunctival inoculation was of lower intensity and duration than that induced subcutaneously, being the differences more evident in young rams. No genital lesions were produced and genital organs and accessory sexual glands were never found infected, being Rev 1 infection restricted to lymph nodes and spleen. Immunostained Rev 1 bacteria were located intracellularly in plasmablasts, dendritic follicular cells and macrophages in the target lymph nodes, in which cellular hyperplasia was the dominant pathological response. Subcutaneous vaccination induced a generalized infection by 2 weeks after vaccination, being then restricted to the prescapular target lymph node. Infection after conjunctival vaccination was less generalized, being restricted essentially to the cranial lymph nodes. Rev 1 infection was fully cleared by 3 months after vaccination in all animals. These results confirm the innocuousness of B. melitensis Rev 1 vaccine in rams.

Rac2 is a major actor of Drosophila resistance to Pseudomonas aeruginosa acting in phagocytic cells.

Pathogen recognition and engulfment by phagocytic cells of the blood cell lineage constitute the first line of defense against invading pathogens. This cellular immune response is conserved throughout evolution and depends strictly on cytoskeletal changes regulated by the RhoGTPases family. Many pathogens have developed toxins modifying RhoGTPases activity to their own benefit. In particular, the Exoenzyme S (ExoS) toxin of the Gram-negative bacteria Pseudomonas aeruginosa is directly injected into the host cell cytoplasm and contains a GAP domain (ExoSGAP) targeting RhoGTPases. Searching for the contribution of each RhoGTPases, Rho1, Rac1, Rac2, Mtl (Mig2-like) and Cdc42 to fly resistance to P. aeruginosa infections, we found that Rac2 is required to resist to P. aeruginosa and to other Gram-negative or Gram-positive bacteria. The Rac2 immune-deficient phenotype is attributable to defective engulfment of pathogens since Rac2-mutant macrophages exhibited strong reduction in the phagocytosis level of both Gram-negative and Gram-positive bacterial particles whereas systemic immune signaling pathways, including Toll, Immune deficiency and Jun kinases, were not affected. Co-expression of Rac2 and ExoSGAP rescued the increased sensitivity to P. aeruginosa observed in ExoSGAP-expressing flies suggesting that Rac2 is the main host factor whose function is inhibited by the GAP domain of the ExoS toxin.