Note added

The "portal spaces" pattern is diverse, some sera display positive bile ducts. Specificities suggestions may be found in PMID 2727702 and PMID 29678444.

ANOTHER RETICULIN PATTERN

Since November 1996 to October 2005, a total of 69815 sera were sent to our laboratory for non-organ specific autoantibodies testing. Antibodies were assayed by an indirect immunofluorescence (IIF) method on rat stomach, renal cortex and liver tissue slides as described before. Throughout the period of study, slides were evaluated on the fluorescence microscopes by the same reader together with a number of training residents.

We were able to recognize a number of reticulin and other unusual patterns of antibodies for which the clinical significance has been only poorly studied. Reticulin patterns R1, R2 and Rs were already well known. Instead of the so-called “adherent cells” pattern we defined a new “variant reticulin” (RV) describing its special features in all three rat tissues (see previous articles). Similarly, we noted that the reticulin pattern of hepatic sinusoid kuppfer cells came together with intravascular clump and other endothelial stainings in different rat tissues. Finally, a new pattern of hepatic sinusoid PMN cells has already been described in another accompanying article as well.

Hereby we present a novel endothelial pattern similar but distinct from RV, which we used to  call “portal area reticulin” or “granular endomysial reticulin”. The endomisium at the gastric circular muscle layer is positive, but less lineal and more granular or discontinuous than R1 or Rs-associated patterns. The myenteric plexus ganglia are positive, also characteristically coarsely granular with maybe a few “ghost” (less intense) spaces. A granular positivity at renal endothelia between the tubules may occur. The staining of portal areas connective tissue is a very specific feature. Sometimes, gastric mucosal granular staining may associate.

Case # 1 - Portal area

Case # 1 - rat kidney



Case # 1 - rat stomach

Case # 1 - rat stomach and ganglia

DETECTION OF NOVEL ANTI NEUTROPHIL CYTOPLASMIC ANTIBODIES (ANCA) IN RAT LIVER

This text is based on the contents of a poster presented at the 25th National Congress of the Spanish Society of Immunology held in Torremolinos in the year 1999. An abstract in Spanish was published at the same event by J Rosique Román and R Alvarez López.

SUMMARY

Introduction: ANCA have recently been shown to be essential for the diagnosis and follow-up of a group of chronic inflammatory disorders. The most useful known targets are proteinase 3 (PR3) for Wegener’s disease or myeloperoxidase (MPO) for microscopic panarteritis nodosa (MPAN). There are a number of other ANCA specificities for which lighter associations have been reported with such autoimmune disorders as lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease or primary sclerosing cholangitis. In clinical practice serum samples are routinely screened for their presence by indirect immunofluorescence (IIF) techniques using ethanol-fixed human neutrophil smears. Little is known about the etiology or build-up of the ANCA immune response.

 

Methods: During the last two and a half years a total of 15000 sera were submitted to our laboratory for the study of non-organ specific autoantibodies. IIF testing was performed through standard procedures on rat stomach, kidney cortex and liver.

Results: 369 patients showed a cytoplasmic staining of polymorphonuclear cells scattered within rat liver sinusoids or less frequently within kidney glomeruli or just inside any vessel. In 158 of these patients a characteristic gastric mucosal granular cytoplasmic staining was also apparent. Other associated findings were: positive ANCA, positive biliary duct granular staining in rat liver, zona pellucida staining in monkey ovary tissue.

Conclusion: there are more ANCA than those currently detectable in routine testing procedures. They be significant for the pathogenesis of ANCA-associated inflammatory disorders or take part in antigen extension pathways. Shared antigenicity between neutrophils and biliary ducts may be relevant for the understanding of primary sclerosing cholangitis-associated ANCA.

INTRODUCTION

Anti-neutrophil cytoplasmic antibodies (ANCA) are a group of autoantibodies that can be detected in human serum samples by indirect immunofluorescence (IIF) testing on human neutrophils. By using ethanol-fixed neutrophils two main patterns have been described, “c” or cytoplasmic, and “p”, perinuclear, which must be differentiated from other true antinuclear antibodies. A number of protein targets have been already reported, such as proteinase 3 (PR3), myeloperoxidase (MPO), lactoferrin, elastase, cathepsin G, bactericidal/permeability-increasing protein (BPI), lysozyme and azurocidin. PR3 antibodies are routinely ordered in clinical practice because of their association with Wegener’s disease, whereas MPO antibodies are useful for the management of microscopic panarteritis nodosa (MPAN) and rapidly progressive glomerulonephritis (RPGN). They are also commonly found in Goodpasture’s glomerulonephritis. The other specificities have less diagnostic value and have been unconsistently associated with lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease (Crohn or ulcerative colitis), primary sclerosing cholangitis or other vasculitides (Churg-Strauss, Takayasu, PAN). A pathogenic mechanism has been suggested by which the neutrophil plays a prominent role by undergoing activation and degranulation and causing damage to “innocent” by-standing tissues. There are also reports that support the possibility of endothelial expression of these autoantigens.

In our setting ANCA are routinely screened through IIF testing on ethanol-fixed neutrophils. Positive samples may undergo further characterization and quantitation with the use of a battery of enzyme immunoassays. It is recommended that the presence of non-organ specific nuclear and cytoplasmic autoantibodies be excluded by other IIF tests, thus helping to differentiate anti-cytoskeleton or anti-ribosomal autoantibodies from c-ANCA patterns, or antinuclear antibodies from p-ANCA. In case there is doubt about the purity of the antigen in any enzyme immunoassay, Western blot or immunoprecipitation studies may be necessary. For clinical routine it is acceptable that an experienced reader be appropriately trained for recognizing the microscopy patterns of the most clinically important specificities but cautious interpretation of results by the clinician is also advisable. Antibody-captured antigen assays may prove to play an essential role for the quantitation of antibodies recognizing conformation-dependent epitopes.

By IIF testing on rat stomach, kidney cortex and liver it is very common that a group of heterophile antibodies are found that recognize the brush border of rat proximal renal tubules. Some of the patients display specially brisk responses that extend to rat liver sinusoids and rat stomach endomysium and are thus reported as Rs reticulin antibody positive.

Zona pellucida autoantibodies have been described and proposed as a mechanism of female infertility. A group of bullous skin diseases have been shown to associate with the presence of autoantibodies directed to squamous epithelial intercellular substances (ICS).

MATERIAL AND METHODS

Patients:

We have computed the last 15000 sera that were sent to our laboratory for non-organ specific autoantibodies or RA testing (approximately after June 1996).

Antibodies assessments:

Non-organ specific antibodies: IIF procedure was performed on rat stomach, renal cortex and liver tissue slides (Biosystems, Barcelona). The initial dilution of sera for screening was 1/40. Bound antibodies were then stained with FITC-conjugated anti-human G,A,M, kappa, lambda antibody.

Other organ specific antibodies were similarly determined by IIF on monkey oesophagus, pancreas or ovary slides (Biosystems, Barcelona), or ethanol-fixed neutrophils (Inova). Sometimes a monkey preabsorbed FITC conjugate was used to minimize nonspecific background staining.

RESULTS

369 patients showed a cytoplasmic staining of polymorphonuclear cells that could be spotted within rat liver sinusoids or less frequently in kidney glomeruli or just inside any vessel. A halo was commonly observed around these cells as if the antigen of interest was diffusing away from its original location.

Of those patients, 40% also showed a granular cytoplasmic pattern of gastric (principal) mucosal cells, sometimes stronger at the luminal side. Similarly strong staining of clumps of secretory material within the glands may also be observed at this site. Most high-titer sera also display a granular staining of biliary ducts in liver portal areas.

This pattern could be seen – with difficulty, because of the high background staining – very commonly in sera with high titer heterophile “brush border” and reticulin Rs antibodies.

The finding of antinuclear, smooth muscle, mitochondrial or reticulin antibodies in the same serum was possible but deemed coincidental.

The strongest sera were also tested in several monkey tissues. Common findings in monkey oesophagus were positive staining for squamous epithelium SIC, or capillary vessel “dots” between muscular bundles. In monkey pancreas we commonly detected enhanced staining of exocrine acinar secretory granules. In monkey ovary tissue the most prominent staining usually was in the vascular spaces aroung tertiary or Graafian follicles or within the zona pellucida around both actively developing or atresic oocytes.

Routine ANCA testing in ethanol- or formalin-fixed neutrophils was typically negative but for a few cases. However, between the neutrophils in the same slides some unidentified amorphous “ghost” round bodies might show a strong positive staining. One of the p-ANCA positive sera was finally diagnosed as having MPO antibodies.

CONCLUSION

A new pattern of antibodies can be defined by IIF testing on rat liver, stomach and kidney which consists of polymorphonuclear cells with a halo around them, gastric mucosal granular cytoplamic and biliary duct staining. These antibodies may also be considered a new type of ANCA.

This immune response might prove to be an extension of one of the commonest “natural” human antibody responses, i.e. that of “rat heterophile” or “brush border” antibodies. It is worth investigating whether this group of antibodies can initiate any type of immune recognition in squamous epithelial SIC, zona pellucida, or endothelia that ultimately lead to more severe or clinically significant chronic inflammatory diseases.

There probably exists an antigen shared between rat neutrophils, gastric mucosa and biliary ducts. If the corresponding antigen can be found in human cells it should be investigated as a target of the primary sclerosing cholangitis-associated immune response.



Case # 1 - rat liver shows biliary duct and halo

Case # 1 - rat liver shows four halos

Case # 1 - rat gastric mucosa shows granular pattern

Case # 2 - rat liver shows neutrophil in detail

Case # 2 - rat gastric mucosa shows granular pattern







CELIAC DISEASE ASSOCIATES WITH TWO DIFFERENT PATTERNS OF ANTIRETICULIN ANTIBODIES

This is an adaptation from the contents of a poster presented at the 24th National Congress of the Spanish Society of Immunology held in Murcia in the year 1998. An abstract in Spanish was published at the same event by J Rosique Román and R Alvarez López.

SUMMARY

Introduction: Rizzetto M and Doniach D (J Clin Pathol 1973 26:841-51. PMID:4587940) reported in 1973 five types of anti-reticulin antibodies (RA) that could be detected in human sera (R1, R2, Rs, Rac and Rkc) mostly through their indirect immunofluorescence (IIF) staining patterns in rat liver, but now other tissues are of common clinical use as well. It had already been established that RA-R1 appear specifically in celiac disease, but the significance of the others remained unclear. In recent times a related specific marker of celiac disease has been described based on testing on primate oesophagus, the IgA antiendomysial antibody (EMA).

Methods: The study includes all sera submitted to our laboratory for study of non-organ specific autoantibodies or RA since January 1996. IIF testing was performed through standard procedures on three rat tissues (stomach, kidney cortex and liver). An ELISA method was used for the measurement of IgA wheat gliadin (AGA) antibodies. IgA antiendomysial antibodies (EMA) were also tested where appropriate.

Results: We describe a pattern of RA antibody different from R1, R2, Rs or Rkc in all three rat tissues. We have found it at 1/160 serum dilution in 44 patients. Of these, 21 (48%) were positive for IgA gliadin antibodies, and 20 (45%) for IgA antiendomysial antibodies.

Conclusion: RA antibodies can be an unexpected finding in routine autoimmunity testing. The pattern of RA antibodies described here is different from R1. If present in high titer it has a definite predictive value for celiac disease, so further diagnostic workup is recommended.

INTRODUCTION

Reticulin antibodies are a very heterogeneous group of antibodies defined by their staining patterns in rodent tissues. Their name comes out of their similarity to some previously described connective tissue fibres, but the term actually encompasses most antibodies to components around renal tubules, muscle fibers, neural or vascular elements, or hepatic sinusoids. Other authors (Storch W B) may describe “antiendothelial” antibodies as a separate group.

RA-R1 make a pattern of coarse reticulin fibre staining around renal tubules, hepatic portal areas and central lobular veins even in low-titer sera. Hepatic sinusoid-associated fibres of characteristic “curly hair” appearance may be observed and is a unique feature. It also stains the stomach submucosal spaces, and the endomysium of smooth muscle fibers. If these are cut transversally a “honeycomb” pattern becomes apparent. These antibodies are specifically associated with untreated celiac disease.

RA-R2 main characteristic is the thinness or delicacy of the fibrils, and their distribution in muscular layers, or blood vessels. They can be seen sparsely within stomach smooth muscle tissue, or within arterioles, thus becoming visible in the kidney or the hepatic portal areas as well. Hepatic central lobular veins may display a fine “radial” pattern.

RA-Rs make the whole staining of hepatic sinusoids, and typically come in association with strong heterophile proximal renal tubular “brush border” and gastric parietal cell antibodies. Honeycombing and granular staining of the myenteric plexus ganglia of the stomach may also occur.

RA-Rkc stain hepatic sinusoid kuppfer cells. Our impression is that this pattern also comes characteristically with “dots” inside the kidney glomeruli. At other vascular spaces we may find “intravascular clumps” or endothelial linings.

MATERIAL AND METHODS

Patients:

From January 1996 to May 1998, a total of 13500 sera were sent to our laboratory for non-organ specific autoantibodies or RA testing.

We scored the following significant significant RA positivities: RA-R1 in 85 patients, RA-R2 in 130 patients, RA-Rs in 37 patients. We found 57 patients that displayed at 1/160 dilution a pattern that stained the vascular endothelia between the renal tubules, making an overall R1-like appearance there, whereas the stomach showed no honeycombing at the circular smooth muscle layer but a positive staining of the myenteric plexus ganglia and other neural elements. Forty-four of these patients were further tested for gliadin and EMA antibodies.

Antibodies assessments:

RA: IIF procedure was performed on rat stomach, renal cortex and liver tissue slides (Biosystems, Barcelona). The initial dilution of sera for screening was 1/40. Bound antibodies were then stained with FITC-conjugated anti-human G,A,M, kappa, lambda antibody.

 

EMA were assessed by IIF testing on monkey oesophagus slides (Biosystems, Barcelona). Sera were diluted 1/10, incubated, washed and finally revealed by FITC-conjugated anti-human A antibody.

Gliadin antibodies: Each Maxisorp (Nunc) microtiter well was sensitized overnight with 1 µg wheat gliadin (Sigma). After blocking with bovine albumin, the wells were incubated with 100 µl of samples at 1/400 dilution. After washing, bound antibodies were revealed with peroxidase-conjugated anti-human IgA and OPD-based color development for 2-3 minutes. For the calculation of  final concentrations a standard calibration curve was constructed by running in parallel 8 serial dilutions of a stored serum, and the relative units of each sample were then calculated out of their color density readings.

Computation of results: the amount of these antibodies in celiac disease rapidly vanishes depending on treatment or adherence to the diet. For the present study we enter the highest recorded value for each patient.

RESULTS

Description of our reticulin antibody “R1-variant”:

In rat liver you can observe staining of central lobular veins endothelia and adjacent sinusoids, as well as a few round cells within the sinusoids.

In rat renal cortex, there is staining of endothelia of (roughly triangular) vascular spaces between the tubules, as well as small intense dots within glomeruli. Some distal tubules may show cytoplasmic staining with the most intense sera.

The rat stomach displays granular fluorescence of the myenteric plexus ganglia between the smooth muscle circular and longitudinal layers, along with “stars” within the circular layer and “worms” between the mucosal glands which might correspond to neural elements too. There is absolutely no honeycombing unless other RA patterns coexist.

Endothelial images can also be seen within the loose connective tissue between the muscularis mucosae and the circular smooth muscle layer.

Associated antibodies

Of 57 patients with RA-R1, 43 were significantly positive for AGA, 14 negative. Of 60 patients with RA-R1, 58 were EMA positive and 2 negative, a fact that may be due to concomitant IgA deficiency.

Of 44 patients with RA-R1-variant, 21 were significantly positive for AGA, of which 19 were EMA positive. Of the remaining 23, only one was EMA positive.

Of a control group of 14 patients with RA-R2, only one was AGA positive, and EMA negative.

CONCLUSION

This might prove to be a novel antibody response pathway associated with celiac disease. Correlation with genetic or diet modifiers may be worth studying.

The incidental finding in a patient of either R1 or R1-variant antibodies should prompt further diagnostic workup aimed at testing more specific markers of celiac disease. The rat tissue IIF test in our setting has so far proved very effective in detecting unsuspected cases of this disease the prevalence of which is much higher than was previously thought.

RA-variant in rat kidney



RA-variant in rat liver









RA-variant in rat stomach

















RA-R1 in rat kidney



RA-R1 in rat liver



 

RA-R1 in rat stomach