Divisions, Laboratories and Service Units:
 
Division of Pathology and Körber-Laboratory for AIDS-Research
(Leider nur in englisch verfügbar!)
 
 
 
 
 
 
 
Figur 1
 
Figur 2
 
 
 
Fig. 1  Benign lymphoepithelial cyst (BLC) of the parotid gland in HIV infection. The in situ hybridziation to detect HIV RNA in combination with immunohistochemistry shows that HIV gene expression (black dot in the lymphoepithelium) inversely correlates with the distribution of TIA-1+ cytotoxic cells (red). 
  
Fig. 2  Lymphoepithelium of a BLC. Cells expressing HIV RNA (black dots) are present in the lymphoepithelium. The immunostaining with antibodies to S-100 protein shows the distribution of dendritic cells (brown). 
  
 
Investigators’ Reports
 

Comparison of HIV-1 Gene Expression from the Effector and the Inductive Sites of the Mucosal Immune System (MIS) 

L. Moncada-Guttierrez, K. Tenner-Racz, K. Evers, B. Raschdorff, M. Dietrich*), P. Racz 

The MIS is of paramount importance in the pathogenesis of HIV-1 infection. The effector site consists of immune-inflammatory cells in the lamina propria and epithelium. The inductive site is composed of the mucosa associated lymphoid tissue (MALT). To date, HIV-1 gene expression within the mucosa has mainly been investigated at the effector sites. The two sites possess differing cellular composition and function, consequently, differences in virus replication can be predicted. To examine the distribution of productively infected cells we immunostained and hybridized gut biopsies with and without MALT (15 and 20 cases, respectively; CDC I-IV C2) and 3 benign lymphoepithelial cysts (BLC) of the parotid gland to an 35S-labeled RNA probe of HIV-1. In 7 cases comparisons with lymph node changes were made. Only 5/20 gut biospies from the effector site  showed a few RNA+ cells in the lamina propria. In none of the cases there were HIV-1+ cells within the epithelium. In contrast, all 15 biopsies containing MALT and the BLCs exhibited high hybridization signals in the germinal centers similar to those seen in the lymph nodes of the same patients. If follicle associated epithelium was present, like in the epithelial lining of the BLCs, HIV-1 preferentially replicated at this site, mainly in CD4+ T cells. In 11 patients with CD4 count, between 9 and 360 infected germinal centers and many CD4 cells were still present in the MALT. We conclude that not the effector site of the MIS, rather the MALT,  including its follicle-associated epithelium is one of the anatomic sites of ongoing HIV replication throughout the spectrum of HIV-1 disease. 
Supported in part by BMBF and the Körber-Foundation 
*) Division of Clinical Medicine 
 

Tonsils as Port of Entry of SIVmac  

P. Racz, K.Tenner-Racz, G. Großschupff, B. Raschdorff, G. Hunsmann*), R. M. Steinman•), C. Stahl-Hennig*) 

Baba et al. (Science, 272:1486, 1996) have succeeded in infecting adult macaques with cell-free SIV orally. They emphasize, that cell-free SIV is significantly more transmissible through the oral route in comparison with the intrarectal route. However, the virus application was not undertaken directly on the tonsils, and morphological examinations of these were not done. Our present study has provided evidence for the first time, that in the oral infection the tonsillar route is possible and the tonsils themselves are of paramount importance. 
Rhesus monekys (Macaca mulatta) were infected via the tonsil with the highly pathogenic macrophage-tropic SIVmac251 grown in monkey peripheral blood mononuclear cells (PBMC) by touching the tonsil regions lightly with a cotton-wool swap soaked with cul-tured medium containing cell-free viruses. The infection of the animals via the tonsils was developed by C. Stahl-Hennig. This is a non-traumatic tonsillar exposure to cell-free SIV. Cotton-wool was soaked with virus and the tonsils lightly  padded  with this three times.
SIV proviral DNA was monitored in PBMC by polymerase chain reaction (PCR) on a regular basis. The cell-associated viral load in PBMCs was determined by a limiting  co-culture technique at the same time points at which PCR analysis was performed. The monkeys  were sacrificed at days 2, 4, and 7 after infection (one animal per time point). Proviral DNA and load of infectious virus were determined using mononuclear cell suspensions from pieces of palatine and lingual tonsils, lymph nodes (cervical, axillary, submandibulary, mesenteric), spleen and thymus.
For in situ hybridization paraffin and cryostat sections were prepared  from the palatine and lingual tonsils, lymph nodes (cervical, submandibular, axillary, paragastric, mesenteric and inguinal), spleen, thymus, bone marrow and gut associated lymphoid tissue (GALT). For analysis of the MALT, samples were taken from the stomach, duodenum, proximal and distal parts of the ileum, colon and rectum. The sections were hybridized with a 35S-labeled single-stranded (antisense) RNA probe of SIVmac239.
Results of these experiments clearly demonstrate that SIV can enter the body through non-traumatized surfaces of tonsils. We have found no virus producing cells in the tonsils at day 2 post infection but SIV DNA+ cells  could be  already detected in the palatine tonsils. However, virus could be isolated from the palatine tonsil at a very low level, whereas all other lymphoid organs tested remained virus isolation negative. This matches with the findings of in situ hybridization showing  that other organs were free of virus nucleic acids.  However, at day 4 after infection many cells replicated SIV (up to 150 SIV RNA+ cells per cutting levels) both in the palatine and the lingual tonsils. The majority of these cells were in the extrafollicular parenchyma and were characterized by a very high hybridization signal (grain counts). In the germinal centers (GC) signs of virus trapping by follicular dendritic cells (FDC) were absent. However, a few GCs already contained virus producing cells. It is important to mention that in these GCs the number of CD8+ lymphocytes was elevated. Occasionally, cells with virus RNA could also be detected in the epithelium. In contrast to the tonsils only 1 or 2 productively infected cells with low grain counts per cutting level were seen in the sinuses of cervical, axillary and paragastric lymph nodes. Other lymphoid organs were free of virus producing cells. Virus isolation by coculture technique revealed a high cell-associated viral load in the palatine tonsil. Also all other lymphoid organs, except for the thymus, were virus isolation positive but with markedly lower viral loads. PCR analysis revealed that the number of SIV DNA+ cells sharply increased in the tonsils. Small amounts of DNA+ cells could be detected in the regional lymph nodes and the spleen.
At day 7 after infection virus replication in the tonsils was more pronounced than that at the previous time points. The number of productively infected cells began to rise in the lymph nodes. They were also seen in the GALT. However, the level of virus production at these sites was significantly lower than that in the tonsils. It should be noted that at this time point of sacrifice the cell-associated virus loads in the blood and the tonsil were similarly high.

The results indicate that tonsils can serve as a port of entry for SIVmac. They also demonstrate that the initial burst of virus replication occurs at the port of entry. Detailed immunohistologic analysis of the host reaction will be continued.
This study is a German-American collaboration. The American investigators are: R. Steinman, and M. Pope.
The American investigators are supported by an NIH-Fogarty International Center Grant. (PA-95-011).
The German investigators are supported by  BMBF  FKZ: 01KI 9484 and  the Körber Foundation.
*) Department of Virology, German Primate Center, Göttingen
•) The Laboratory of Cellular Physiology and Immunology, Rockefeller University, New  York, USA
 
 

Atrophic Germinal Centers of Lymph Nodes Resembling those of Hyalin-vascular Type of Castleman’s Disease Contain High Amounts of HIV-1 RNA

P. Racz, I. Karstens, B. Raschdorff, M. Dietrich*), K.Tenner-Racz

During the last decade it has been well documented that follicular dendritic cells (FDC) capture and retain HIV. Thus, hyperplastic follicles represent important virus reservoirs. It has also been suggested that in the late phase of the disease the virus trapping capability of FDC is lost. To gain information about the role of FDC in the late stage of disease we analyzed small atrophic and hypo-cellular germinal centers in 11 lymph nodes with changes resembling those seen in hyalin vascular type of Castleman’s disease. In 9/11 cases biopsies were taken 3-17 months before death. 5/11 patients had Kaposi’s sarcoma. Paraffin and cryostate sections were evaluated  for FDC, p24 of HIV-1, sIgD, sIgM and CD4+ cells. To detect HIV RNA in situ hybridization was performed on  immunostained paraffin section with the use of an 35S-labeled antisense probe. The majority of the GCs was surrounded by a broad
mantle zone consisting of concentrically arranged IgD+, IgM+ small lymphocytes. In 3 cases these cells infiltrated the paracortical areas. The GCs were rich in FDC, the dendrites were thickened. In association with FDC large amounts of HIV RNA signals were always present. Similarly, in these follicles strong reaction with p24 was noted. Productively infected CD4+ T cells were also present.
Conclusions:  FDCs in atrophic germinal centers do not loose the capability to trap and retain HIV-1. Even at the late stage of the disease they represent an HIV depository as long as they are present.
Supported in part by BMBF and the Körber Foundation
*) Division of Clinical Medicine
 


 
Staff 

Prof. Dr. Paul Racz 
Dr. Wilhelm Büngener 
Dr. Gertrud Helling-Giese 
Dr. Klara Tenner-Racz 
  

Visiting Scientists 

Prof. Dr Françoise Barré-Sinoussi, Institut Pasteur, Paris 
Prof. Dr. Michel Huerre, Institut Pasteur, Paris 
Prof. Dr. Ralf M. Steinman, Rockefeller University, New York 
Prof. Dr. Ashley T. Haase, University of Minnesota, Minneapolis, USA 
Dr. Marten Schutten, Erasmus University, Rotterdam 
 

Doctoral Students 

Kirsten Arndt 
Eva Kahn 
Ines Karstens 
Liliana Moncada-Guttierrez 
Heidemarie Schmidt 
Felicitas van Vloten 
 

Support Staff 

Miriam Dücker 
Kirsten Evers 
Gudrun Großschupff 
Birgit Raschdorff 
Güler Yilderim