Comprehensive assessment of HIV target cells in the distal human gut suggests increasing HIV susceptibility toward the anus.

BACKGROUND: Prevention of rectal HIV transmission is a high-priority goal for vaccines and topical microbicides because a large fraction of HIV transmissions occurs rectally. Yet, little is known about the specific target-cell milieu in the human rectum other than inferences made from the colon. METHODS: We conducted a comprehensive comparative in situ fluorescence study of HIV target cells (CCR5-expressing T cells, macrophages, and putative dendritic cells) at 4 and 30 cm proximal of the anal canal in 29 healthy individuals, using computerized analysis of digitized combination stains. RESULTS: Most strikingly, we find that more than 3 times as many CD68 macrophages express the HIV coreceptor CCR5 in the rectum than in the colon (P = 0.0001), and as such rectal macrophages seem biologically closer to the HIV-susceptible CCR5 phenotype in the vagina than the mostly HIV-resistant CCR5 phenotype in the colon. Putative CD209 dendritic cells are generally enriched in the colon compared with the rectum (P = 0.0004), though their CCR5 expression levels are similar in both compartments. CD3 T-cell densities and CCR5 expression levels are comparable in the colon and rectum. CONCLUSIONS: Our study establishes the target-cell environment for HIV infection in the human distal gut and demonstrates in general terms that the colon and rectum are immunologically distinct anatomical compartments. Greater expression of CCR5 on rectal macrophages suggests that the most distal sections of the gut may be especially vulnerable to HIV infection. Our findings also emphasize that caution should be exercised when extrapolating data obtained from colon tissues to the rectum.

Expression of chemokines and chemokine receptors during human renal transplant rejection.

Infiltration of renal allografts by leukocytes is a hallmark of acute transplant rejection. Chemokines attract leukocytes bearing specific chemokine receptors, and the specific leukocyte chemokine receptor phenotype is associated with types of immune responses, ie, T helper subtype 1 (Th1; CXC chemokine receptor 3 [CXCR3], CC chemokine receptor 5 [CCR5]) versus Th2 (CCR3, CCR4, CCR8). We studied the expression of the chemokine monocyte chemoattractant protein-1 and the chemokine receptors CCR2B and CXCR4 messenger RNA (mRNA) by in situ hybridization, as well as the chemokine receptors Duffy antigen receptor for chemokines (DARC) and CCR5 protein by immunohistochemistry in renal biopsy specimens with acute cellular rejection (n = 12) and acute vascular rejection (n = 8), transplant nephrectomy specimens (n = 6), and normal areas of tumor nephrectomy specimens (n = 5). CC chemokines and CC chemokine receptor mRNA expression were evaluated by ribonuclease protection assay in specimens from four transplant nephrectomies and one tumor nephrectomy. Upregulation of mRNAs for the chemokines, interferon-inducible protein-10 (IP-10); regulated on activation normal T-cell expressed and secreted; macrophage inflammatory protein-1alpha (MIP-1alpha); MIP-1beta; and lymphotactin, as well as the chemokine receptors, CCR2 and CCR5, were documented during allograft rejection. CCR1 mRNA was detectable in both allografts and controls, but CCR3 and CCR8 were absent. The number of CXCR4, CCR5, and CCR2B mRNAs expressing leukocytes and DARC-positive vessels increased during rejection episodes. CXCR4 mRNA was the most widely expressed. Leukocytes in diffuse interstitial infiltrates were mainly CCR5 positive, but in areas in which leukocytes formed nodular aggregates of infiltrating cells, the number of CCR5-positive cells was low. Instead, leukocytes in these nodular aggregates mainly expressed CXCR4. DARC was expressed on peritubular capillaries, where it was upregulated in areas of interstitial infiltration. Induction of chemokines during renal allograft rejection is accompanied by infiltration of leukocytes bearing the respective chemokine receptors. The upregulation of the CXCR3 ligand IP-10, as well as CCR5 and its ligands, in the absence of CCR3 and CCR8 is indicative that renal allograft rejection is primarily the result of a Th1-type immune response.

Differential expression of cyclin-dependent kinase inhibitors in human glomerular disease: role in podocyte proliferation and maturation.

BACKGROUND: Normal human podocytes are terminally differentiated and quiescent cells. It is not known why podocytes fail to proliferate in response to most forms of injury. Proliferation is regulated by cell cycle proteins and their inhibitors. The Cip/Kip family of cyclin-dependent kinase (CDK) inhibitors (p21, p27, p57) in general prevent proliferation by inhibiting cyclin-CDK complexes. In the current study, we determined the expression and possible role of specific CDK inhibitors in podocyte proliferation in human disease characterized by podocyte injury. METHODS: Immunostaining was performed for the CDK inhibitors p21, p27, and p57 and the proliferation marker Ki-67 on renal biopsies from patients with minimal change disease (MCD; N = 6), membranous glomerulopathy (MGN; N = 19), cellular variant of focal segmental glomerulosclerosis (FSGS; N = 12), collapsing glomerulopathy (CG; N = 9), and HIV-associated nephropathy (HIVAN; N = 16). Adult nephrectomy specimens without evidence of glomerular disease served as controls (N = 9). RESULTS: Normal quiescent podocytes express p27 and p57, but not p21. In diseases without podocyte proliferation (MCD, MGN), p21, p27, and p57 expression did not change. In contrast, there was a uniform decrease in p27 and p57 immunostaining in diseases with podocyte proliferation (cellular FSGS, CG, and HIVAN). This was accompanied by the de novo expression of p21 in podocytes. CONCLUSIONS: Our results show that podocyte quiescence may require the presence of the CDK inhibitors p27 and p57. In human glomerular diseases, a decrease in p27 and p57 may be permissive for the altered proliferative podocyte phenotype. p21 may have a multifactorial role in podocyte cell cycle regulation.

Abnormal expression of the cystic fibrosis transmembrane regulator in chronic sinusitis in cystic fibrosis and non-cystic fibrosis patients.

Cystic fibrosis (CF) patients commonly suffer from chronic sinusitis. Mutations of a single gene, the cystic fibrosis transmembrane conductance regulator (CFTR) gene, have been associated with CF. Functional CFTR protein is localized to the apical cell membrane, while dysfunctional CFTR is commonly found in the cytoplasm. We undertook a preliminary immunocytochemical study of CFTR subcellular localization in CF and non-CF pediatric and adult patients using a newly developed murine monoclonal antibody, TAM. Immunostaining was evaluated for subcellular localization (cytoplasmic versus membranous) and for epithelial layer (basal versus luminal). Analysis of the predominant CFTR distribution patterns demonstrated significant differences in adult versus pediatric groups independent of whether the latter were CF or non-CF (p<.0001 and p<.008, respectively), and no significant difference between the 2 pediatric groups (p = .70). This suggests that the pathophysiology of pediatric sinusitis differs from that of adult sinusitis at the level of secretion production.

Validation of the S-phase specificity of histone (H3) in situ hybridization in normal and malignant cells.

Several different methods of measuring proliferation indices have been developed, including measurements of cellular DNA content (flow cytometry), S-phase incorporation of thymidine analogues into DNA (e.g., tritiated thymidine and 5'-bromodeoxyuridine), and immunostaining of cell cycle-restricted proteins (e.g., Ki-67 antigen and PCNA). Theoretical and practical problems with each method have made it difficult to compare absolute proliferation rates among cells of different lineages and degrees of malignancy. More recently, in situ hybridization (ISH) for histone 3 (H3) mRNA has been introduced. We used a double labeling method for comparing H3 mRNA expression and S-phase incorporation of 5'-bromodeoxyuridine (BrdU) to determine if H3 mRNA expression was tightly associated with S-phase in a variety of malignant and nontransformed cell types. In addition, labeling results were compared in methacarn- and formalin-fixed tissues to extend the potential usefulness of H3 ISH, using a postfixation technique for the alcohol-fixed specimens. As expected for a cumulative marker, variation was noted in the percentage of the BrdU-positive cells double labeled with H3 ISH (53-89%), depending on cell type and length of BrdU incubation. In contrast, the percentage of the H3 ISH-positive cell population double labeled for BrdU was independent of the cell type of BrdU incubation time (mean 78%). Similarly, a consistent percentage of H3 ISH-positive cell populations was double labeled for BrdU in normal tissues (mean 97%). These findings support a well-conserved timing mechanism for H3 mRNA expression and DNA replication. We conclude that H3 ISH is an extremely accurate technique for assessment of S-phase cell proliferation indices.