The monoclonal antibody Ki-67 reacts with a human nuclear cell proliferation-associated antigen that is expressed in all active parts of the cell cycle. Recently we have raised monoclonal antibodies, MIB 1-3, against recombinant parts of the Ki-67 antigen. These antibodies are true Ki-67 equivalents, as demonstrated by immunostaining of fresh specimens, biochemistry, and molecular biological techniques. Formalin-fixed, paraffin-embedded sections routinely processed for immunohistochemistry failed to stain for Ki-67 and MIB 2. Antibodies MIB 1 and MIB 3 labelled mitotic figures, while non-mitotic proliferating cells were negative under these conditions. However, when dewaxed microwave oven-processed paraffin sections of formalin-fixed tissues were used, MIB 1 and MIB 3 gave strong nuclear staining of those cells presumed to proliferate under a variety of normal and neoplastic conditions. Moreover, routine decalcification or depigmentation techniques did not alter the immunoreactivity of MIB 1 and MIB 3 with microwave-processed paraffin sections. This method is highly reproducible, easy to perform at low cost, and no additional technical skill is needed because after microwave treatment just routine immunohistochemical methods are used. Since we have successfully applied this new method to sections obtained from paraffin blocks stored for a long time (in one case more than 60 years), the assessment of cell kinetics through the detection of Ki-67 antigen is now possible on archival material collected in histopathology departments all over the world.

Abstract. The antigen defined by mAb Ki-67 is a human nuclear protein the expression of which is strictly associated with cell proliferation and which is widely used in routine pathology as a "proliferation marker" to measure the growth fraction of cells in human tumors. Ki-67 detects a double band with apparent molecular weights of 395 and 345 kD in immunoblots of proteins from proliferating cells. We cloned and sequenced the full length eDNA, identified two differentially spliced isoforms of mRNA with open reading frames of 9,768 and 8,688 bp encoding for this cell proliferation-associated protein with calculated molecular weights of 358,761 D and 319,508 D, respectively. New mAbs against a bacterially expressed part and a synthetic polypeptide deduced from the isolated eDNA react with the native Ki-67 antigen, thus providing a circle of evidence that we have cloned the authentic Ki-67 antigen eDNA. The central part of the Ki-67 antigen eDNA contains a large 6,845-bp exon with 16 tandemly repeated 366-bp elements, the "Ki-67 repeats", each including a highly conserved new motif of 66 bp, the "Ki-67 motif", which encodes for the epitope detected by Ki-67. Computer analysis of the nucleic acid and the deduced amino acid sequence of the Ki-67 antigen confirmed that the cDNA encodes for a nuclear and short-lived protein without any significant homology to known sequences. Ki-67 antigen-specific antisense oligonucleotides inhibit the proliferation of IM-9 cell line cells, indicating that the Ki-67 antigen may be an absolute requirement for maintaining cell proliferation. We conclude that the Ki-67 antigen defines a new category of cell cycleassociated nuclear nonhistone proteins.

The CXC chemokines interleukin-8 and GRO/melanoma growth-stimulatory activity (GRO-alpha) are potent activators of neutrophils and lymphocytes, but also stimulate growth and differentiation of nonhematopoietic cells like keratinocytes, fibroblasts, and melanocytes. High mRNA and protein levels have been detected in psoriatic epidermis. Chemokine activation of target cells is mediated by specific receptors and two CXC receptors have been described with similar affinity for interleukin-8 but different affinities for GRO-alpha. In this study, we examined the expression of both CXCR1 and CXCR2 in psoriatic tissue, identifying the target cells of chemokine activation in psoriasis. By immunohistochemistry and in situ hybridization, as confirmed by northern blot analysis and reverse transcriptase polymerase chain reaction, we could detect expression of the CXCR2 in suprabasal lesional psoriatic keratinocytes but not in healthy skin. The CXCR1 could not be localized in psoriatic keratinocytes with immunohistochemistry and in situ hybridization, but infiltrating cells in the dermal compartment expressed both types of receptors. These data suggest that in addition to neutrophil activation by both CXCR1 and CXCR2, activation of keratinocytes mediated by CXCR2 could contribute to the characteristic epidermal changes observed in psoriasis.

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Aims-To elucidate the fine specificities of the antibodies MIB 1 and MIB 3 and of additional monoclonal antibodies which also recognise the Ki-67 protein (MIB 5, IND.64, First studies showed that the epitopes of MIB 1, MIB 3, and Ki-676 as well as that of MIB 5 (unpublished results) are located on an identical stretch of 20 amino acids within the Ki-67 protein. This sequence represents a part of the highly conserved 22 amino acid element called "Ki-67 motif'. In the Ki-67 protein it appears 16 times with a homology between 72% and 100% with regard to the amino acid sequence ( fig 1A).'0 Because of the importance of estimating the Ki-67 growth fraction, the aim of our study was to elucidate the fine specificities of antibodies directed against the Ki-67 protein within the 22 amino acid sequence mentioned above, and to compare the epitopes recognised by the antibodies MIB 1, 3, 5, Ki-67, and JG-67-2all and the recently described monoclonal antibody IND.64,12 which represents an additional Ki-67 equivalent antibody.'3 Methods All amino acid sequences mentioned are written in the one letter code from the N-terminus to the C-terminus (from left to right).Antibodies MIB 1, 2, 3, and 5, Ki-67 and IND.64 were generated as described' 6 712; JG- Epi 6 1A and B) were synthesised on a Beckman DNA-SM Synthesizer (Beckman, Munich, Germany) with additional bases on the 5' and 3' end to form restriction sites after hybridisation. The corresponding sense and antisense strands were hybridised by heating for 10 minutes to 95°C and slowly cooling down to room temperature. The double strand fragments were separated on nondenaturing PAGE and eluted from the gel by the "crush and soak" method.'5 The purified fragments were cloned between the Bgl II and Sal 1 site of the pAX 4a + vector and transformed into E coli DH5aIQ.All resulting plasmids carried a gene which encodes for a fusion protein containing bacterial fl-galactosidase and the respective part of the Ki-67 protein.