Journal: Comparative Medicine

Article Title: Chlamydia muridarum Associated Pulmonary and Urogenital Disease and Pathology in a Colony of Enzootically Infected Il12rb2 Deficient and Stat1 Knockout Mice

doi: 10.30802/aalas-cm-24-000002

Figure Lengend Snippet: Figure 1. Cm associated bronchopneumonia, lung, Il12rb2KO mice. (A) Mild to moderate multifocal peribronchiolar and perivascular bron- chopneumonia in a clinically affected female mouse. HE, subgross. (B) Lymphoplasmacytic and histiocytic bronchopneumonia noted boxed in A, with intraepithelial Cm inclusions. HE, 10×. (C) Higher magnification of Cm inclusions noted in hatched box in B (arrows). HE, 40×. (D) Cm signal in bronchiolar epithelial cells within inflammatory lesions noted on HE (arrow, brown staining). IHC for Cm MOMP, subgross. (E) Cm in bronchiolar epithelial cells (brown staining). IHC for Cm MOMP, 10×. (F) Cm nucleic acid in bronchiolar epithelial cell of a clinically normal male Il12rb2KO mouse (red staining). ISH for Cm RNA. (G) Peribronchiolar and perivascular T and B cell infiltrates surrounding Cm positive epithelial cells (CD3+ brown, B220+ red). Double staining IHC for CD3 and B220, 2×. (H) Higher magnification of boxed area in G (CD3+ brown, B220+ red). Double staining IHC for CD3 and B220, 20× (I) Further differentiation of subpopulations of CD3+ cells in same area as in H (CD4+ red, CD8+ brown). Double staining IHC for CD4 and CD8, 20×.

Article Snippet: After deparaffinization and heat-induced epitope retrieval in a citrate buffer at pH 6.0, the primary antibody against chlamydial MOMP (NB100-65054, Novus Biologicals, Centennial, CO) was applied at a dilution of 1:500.

Techniques: Staining, Double Staining

Journal: Comparative Medicine

Article Title: Chlamydia muridarum Associated Pulmonary and Urogenital Disease and Pathology in a Colony of Enzootically Infected Il12rb2 Deficient and Stat1 Knockout Mice

doi: 10.30802/aalas-cm-24-000002

Figure Lengend Snippet: Figure 2. Reactive GALT hyperplasia and Cm colonization in the gastrointestinal tract. (A) GALT hyperplasia with prominent germinal centers, cecum, male STAT1KO mouse. HE, subgross. Inset: Cm signal in surface cecal epithelial cells (brown staining). IHC for Cm MOMP, 10×. (B) His- tologically normal colon from a clinically affected female Il12rb2KO mouse. HE, 10×. Inset: Cm signal in surface colonic epithelial cells (brown staining). IHC for Cm MOMP, 10×. (C) Male STAT1KO mouse, minimal to mild lymphoplasmacytic, histiocytic, and neutrophilic typhlitis, with Cm inclusion (arrow), cecum. HE, 20×. (D) Female STAT1KO mouse, Cm signal within surface epithelial cells at the gastric limiting ridge (brown staining). IHC for Cm MOMP, 4×.

Article Snippet: After deparaffinization and heat-induced epitope retrieval in a citrate buffer at pH 6.0, the primary antibody against chlamydial MOMP (NB100-65054, Novus Biologicals, Centennial, CO) was applied at a dilution of 1:500.

Techniques: Staining

Journal: Comparative Medicine

Article Title: Chlamydia muridarum Associated Pulmonary and Urogenital Disease and Pathology in a Colony of Enzootically Infected Il12rb2 Deficient and Stat1 Knockout Mice

doi: 10.30802/aalas-cm-24-000002

Figure Lengend Snippet: Figure 5. Urogenital lesions, bladder and female reproductive tract, female STAT1KO mouse. (A) Severe multifocal to coalescing ureteritis with intraurothelial Cm inclusions and hyaline droplets; higher magnification inset with Cm inclusion (arrow, 10×). Left ureter. HE, subgross. (B) Severe, diffuse cystitis with occasional intraurothelial Cm inclusions (IHC for Cm MOMP; inset, 20×), urothelial cell necrosis, and adjacent lym- phoid follicular hyperplasia. Bladder. HE, subgross. (C) Severe ovarian parenchymal atrophy with multifocal to coalescing periovarian steatitis and myositis. Left ovary. HE, subgross.

Article Snippet: After deparaffinization and heat-induced epitope retrieval in a citrate buffer at pH 6.0, the primary antibody against chlamydial MOMP (NB100-65054, Novus Biologicals, Centennial, CO) was applied at a dilution of 1:500.

Techniques:

Journal: Comparative Medicine

Article Title: Chlamydia muridarum Associated Pulmonary and Urogenital Disease and Pathology in a Colony of Enzootically Infected Il12rb2 Deficient and Stat1 Knockout Mice

doi: 10.30802/aalas-cm-24-000002

Figure Lengend Snippet: Figure 4. Bilateral hydronephrosis, and Cm-associated unilateral urothelial papilloma, kidneys, female STAT1KO mouse. (A) Moderate hy- dronephrosis with locally extensive parenchymal atrophy, right kidney. HE, subgross. (B) Medullary and cortical tubular degeneration, mild multifocal interstitial nephritis, right kidney. HE, 10×. (C) Severe pyonephritis (neutrophilic inflammation depicted in inset. HE, 10×) with diffuse parenchymal atrophy and urothelial papilloma arising from renal pelvis (bracket), right kidney. HE, subgross. (D) Severe multifocal to coalescing pyonephritis and parenchymal atrophy, indicated by dashed box in C, left kidney. HE, 5×. (E) Severe multifocal to coalescing inflammation and intraurothelial Cm inclusions (boxed area from C), seen only in the urothelium of the papilloma (higher magnification inset); normal urothelial cells without Cm inclusions at top right. Urothelial papilloma; left kidney. HE, 10×. (F) Cm signal in urothelial cells lining the urothelial papilloma, with no signal seen in the normal urothelial cells at top right, left kidney. IHC for Cm MOMP, 10×.

Article Snippet: After deparaffinization and heat-induced epitope retrieval in a citrate buffer at pH 6.0, the primary antibody against chlamydial MOMP (NB100-65054, Novus Biologicals, Centennial, CO) was applied at a dilution of 1:500.

Techniques:

Journal: bioRxiv

Article Title: Reemergence of the Murine Bacterial Pathogen Chlamydia muridarum in Laboratory Mouse Colonies

doi: 10.1101/2022.05.16.491822

Figure Lengend Snippet: Representative histopathology of the lung and cecum from a 1.5-year-old, female, C57Bl/6/Foxp3 CreER R26 tdTomato mouse. A. Multifocally, peribronchiolar and perivascular spaces are infiltrated by small to moderate clusters of lymphocytes and histiocytes (scale bar = 500μm). B. High magnification field shows lymphocytic and histiocytic infiltrates around a bronchiole (scale bar = 100μm). C. IHC of the lung demonstrating focal detection of chlamydial MOMP antigen in bronchiolar epithelial cells (scale bars = 100μm; 20 μm - brown staining in inset). D. Representative section of normal mucosa and gut-associated lymphoid tissue (GALT) in the cecum with low numbers of luminal T. muris (arrowheads; scale bars = 100μm). E. IHC of the cecum demonstrating detection of intracytoplasmic chlamydial inclusions in surface epithelial cells (arrowhead; scale bar = 20 μm). F. ISH demonstrates positive staining (red) for Cm mRNA within the cecal epithelium (arrowhead) and lumen (arrow; scale bar = 20 μm).

Article Snippet: Following deparaffinization and heat-induced epitope retrieval in a citrate buffer at pH 6.0, the primary antibody against Chlamydia MOMP (NB100-65054, Novus Biologicals, Centennial, CO) was applied at a dilution of 1:500.

Techniques: Histopathology, Staining

Journal: bioRxiv

Article Title: Reemergence of the Murine Bacterial Pathogen Chlamydia muridarum in Laboratory Mouse Colonies

doi: 10.1101/2022.05.16.491822

Figure Lengend Snippet: Histopathology of the lung and large intestine from a 1-year-old, female, NSG mouse. A. Representative airway demonstrating a bronchiolar and alveolar inflammation characterized by luminal neutrophilic infiltration mixed with necrotic debris and proteinaceous material. Multifocally, bronchiolar epithelial cells exhibit intracytoplasmic clear vacuoles with pale-basophilic structures compatible with Chlamydial inclusions (arrowhead). Peribronchiolar and alveolar space is infiltrated with moderate numbers of macrophages and neutrophils intermixed with reactive fibroblasts (scale bar = 20 μm). B. IHC of the lung demonstrating detection of chlamydial MOMP antigen in bronchiolar epithelial cells (arrowhead; brown staining) and areas with peribronchiolar inflammation (arrow, scale bar = 20 μm). C. ISH demonstrates positive staining (red) for Cm mRNA in bronchiolar epithelial cells (arrowhead) and areas of peribronchiolar inflammation (arrow; scale bar = 20 μm). D. Representative H&E-stained section of a normal cecal wall (scale bars = 100μm). E. High magnification field demonstrates ISH signal (red staining) in the cecal epithelium (arrow) and lumen (arrowhead; scale bar = 20μm). F. IHC of descending colon demonstrating detection of intracytoplasmic chlamydial MOMP antigen in surface epithelial cells (inset - brown staining; scale bars = 20 −200μm).

Article Snippet: Following deparaffinization and heat-induced epitope retrieval in a citrate buffer at pH 6.0, the primary antibody against Chlamydia MOMP (NB100-65054, Novus Biologicals, Centennial, CO) was applied at a dilution of 1:500.

Techniques: Histopathology, Staining

Journal: bioRxiv

Article Title: Reemergence of the Murine Bacterial Pathogen Chlamydia muridarum in Laboratory Mouse Colonies

doi: 10.1101/2022.05.16.491822

Figure Lengend Snippet: Fluorescent images of Chlamydia -infected cells. HeLa 229 cells were infected with Chlamydia spp. isolated with an NSG mouse cecum sample. At 30 hours post-infection, cells were fixed and stained with FITC-labeled anti -Chlamydia spp. antibody (green) and analyzed using an EVOS™ FL Auto Imaging fluorescent microscope (Life Technologies).

Article Snippet: Following deparaffinization and heat-induced epitope retrieval in a citrate buffer at pH 6.0, the primary antibody against Chlamydia MOMP (NB100-65054, Novus Biologicals, Centennial, CO) was applied at a dilution of 1:500.

Techniques: Infection, Isolation, Staining, Labeling, Imaging, Microscopy

Journal: PLoS Pathogens

Article Title: Attachment and Entry of Chlamydia Have Distinct Requirements for Host Protein Disulfide Isomerase

doi: 10.1371/journal.ppat.1000357

Figure Lengend Snippet: CHO6 cells were transfected with vectors expressing wildtype PDI (CHO6+PDI) or an enzymatic mutant of PDI lacking active site cysteine residues (CHO6+PDI-4CS). 48 h after transfection, cells were infected with Chlamydia for subsequent attachment and entry analysis. Cells were fixed and evaluated by immunofluorescence, bacteria are green and counter staining shown with Evans blue. (A) Bacterial attachment was analyzed 1 h post-infection. Bacterial attachment was recovered in CHO6+PDI as well as in CHO6+PDI-4CS, indicating that PDI enzymatic activity is not necessary for bacterial attachment. (B) The number of bacteria attached to cells was determined by quantification, the number of bacteria associated with each cell in eight separate fields of view containing at least ten cells. Error bars indicate standard of deviation (STDEV). (C) 24 h after infection Chlamydia infectivity was evaluated. Infectivity was restored in CHO6+PDI. No productive infection was observed in CHO6 cells or in CHO6+PDI-4CS. In CHO6+PDI-4CS, the bacteria remained persistently attached to the cell, indicative of the requirement for PDI enzymatic activity for bacterial entry. (D) Infection was quantified by counting the number of inclusions per field of view in eight separate fields of view containing at least ten cells. Error bars indicate the STDEV. (E) Cells were incubated at 37°C for 2 h to allow for bacterial entry. Entry was analyzed by comparing the total number of cell-associated bacteria for staining of permeabilized cells (Total) and the number of extracellular bacteria determined by staining unpermeabilized cells (Extracellular). Bacterial entry was recovered in CHO6 cells expressing PDI (CHO6+PDI) but not in CHO6 cells expressing enzymatically nonfunctional PDI (CHO6+PDI-4CS). (F) Percent internalization represents 1 minus the number of extracellular bacteria divided by the total number of bacteria multiplied by 100. The number of extracellular and total bacteria was determined by quantifying the number of bacteria associated with each cell per field of view in eight separate fields of view containing at least ten cells. Error bars indicate the STDEV.

Article Snippet: Blocking solution was removed and coverslips were incubated 1 h with mouse anti- Chlamydia MOMP antibody for C. trachomatis or anti- Chlamydia LPS antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for C. psittaci and then washed with HBSS.

Techniques: Transfection, Expressing, Mutagenesis, Infection, Immunofluorescence, Bacteria, Staining, Activity Assay, Standard Deviation, Incubation

Journal: PLoS Pathogens

Article Title: Attachment and Entry of Chlamydia Have Distinct Requirements for Host Protein Disulfide Isomerase

doi: 10.1371/journal.ppat.1000357

Figure Lengend Snippet: (A) Chlamydia attachment to CHOK1 was evaluated by immunofluorescence staining of bacteria (green) 1 h after infection and counter staining with Evans blue. Cells were infected and then maintained in media containing 0 mM or 3 mM bacitracin. Similar levels of bacterial attachment were observed with and without bacitracin. (B) Entry was examined after a 2 h incubation at 37°C. Entry was analyzed by comparing the total number of cell-associated bacteria (permeabilized) and the number of extracellular bacteria (unpermeabilized). 3 mM bacitracin inhibited bacterial entry. (C) The effect of bacitracin on Chlamydia attachment and infection was quantified and is shown as the % attachment and infection of cells not treated with bacitracin. Attachment was analyzed 1 h post-infection and infection was evaluated 24 h post-infection. (D) The effect of bacitracin on Chlamydia development was examined by evaluating cells 24 h post-infection that had not been treated with bacitracin (a′), been treated with 3 mM bacitracin for 20 min prior to infection (b′), been treated with bacitracin for the first 8 h of infection (c′), or been treated with bacitracin only for the last 16 h of the 24 h infection (d′). In all cases the development of bacteria-containing vacuoles was seen, indicative of a normal infection.

Article Snippet: Blocking solution was removed and coverslips were incubated 1 h with mouse anti- Chlamydia MOMP antibody for C. trachomatis or anti- Chlamydia LPS antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for C. psittaci and then washed with HBSS.

Techniques: Immunofluorescence, Staining, Bacteria, Infection, Incubation

Journal: PLoS Pathogens

Article Title: Attachment and Entry of Chlamydia Have Distinct Requirements for Host Protein Disulfide Isomerase

doi: 10.1371/journal.ppat.1000357

Figure Lengend Snippet: (A) CHO6 cells transfected with a vector expressing PDI with a gpi-anchor (PDI-gpi) are marked with a white arrow, untransfected cells are indicated by a yellow arrow. Cells were fixed and stained for PDI using PDI-specific antibody. Expression of PDI-gpi led to a large amount of PDI localized at the cell surface. (B) Cell survival was analyzed 72 h after DT treatment. CHOK1 cells were extremely sensitive to toxin, whereas CHO6 cells were relatively resistant. Expression of PDI-gpi in CHOK1 or CHO6 cells did not alter toxin sensitivity, indicative of an inability of PDI-gpi to properly interact with other cell surface proteins. (C) Chlamydia attachment to CHOK1 and CHO6+PDI-gpi was analyzed, bacteria are shown in green, and PDI is stained red. There was no bacterial attachment to CHO6+PDI-gpi.

Article Snippet: Blocking solution was removed and coverslips were incubated 1 h with mouse anti- Chlamydia MOMP antibody for C. trachomatis or anti- Chlamydia LPS antibody (Santa Cruz Biotechnology, Santa Cruz, CA) for C. psittaci and then washed with HBSS.

Techniques: Transfection, Plasmid Preparation, Expressing, Staining, Bacteria