In situ hybridization, in situ transcription, and in situ polymerase chain reaction.

In situ hybridization, in situ transcription, and in situ polymerase chain reaction (PCR) are techniques used to detect DNA and RNA sequences within a cell or tissue structure. These three in situ methodologies employ the principles of recombinant DNA to form double-stranded hybrids of DNA-DNA, DNA-RNA, or RNA-RNA. The essence of in situ hybridization (ISH) is the hybridization of a labeled probe to a complementary target sequence, whereas in situ transcription (IST) is the synthesis of complementary DNA incorporating a label directly on the target DNA or RNA within a cell or tissue. In the case of in situ PCR (ISPCR), it is the repeated in situ duplication of both the sense and antisense strands of DNA to increase the number of copies of the target sequence. ISH, IST, and ISPCR each have their advantages and disadvantages. The purpose of this chapter is to address in situ considerations required of these techniques, emphasizing tissue fixation, pre-hybridization steps, DNA probes, RNA probes, oligoprobes, and probe labeling. Five successfully used protocols are presented as examples. Any given nucleotide target sequence may have its own unique set of optimum conditions, thus requiring some adjustment in the hands of the user.

Enzyme histochemical studies of membrane proteases in rat subfornical organ.

Localization of membrane proteases glutamyl aminopeptidase (EAP), microsomal alanyl aminopeptidase (mAAP), dipeptidyl peptidase IV (DPP IV) and gamma-glutamyl transpeptidase (gamma-GTP) were studied in vessels of the rat subfornical organ (SFO), ependyma which cover the surface of the SFO, and adjacent brain structures. Results of enzyme histochemical reactions showed strong activity for EAP, mAAP, and gamma-GTP, but absence of DPP IV in microvessels of SFO. The ependyma which cover the SFO was positive for gamma-GTP, but negative for other studied proteases. Our results showed that the spectrum of enzymes in the majority of the vessels of SFO is similar to that of the microvessels of the adjacent brain tissue which were positive for EAP, mAAP, and gamma-GTP, but negative for DPP IV. The relative intensity of the enzyme reactions in vessels varied from central to lateral locations in the SFO and the adjacent brain tissue. There was also a difference in the relative reaction intensity from one enzyme to the other. The presence and heterogeneous distribution of the enzymes are consistent with the hypothesis that membrane proteases of the microvascular endothelium constitute an enzyme-barrier between blood and parenchyma of the SFO and between blood and brain tissue, and may be involved in metabolism or modulation of various peptides when they contact the plasma membrane of the endothelial cells of the vessels.

Species differences in the distribution of gamma-glutamyl transpeptidase in choroid plexus of lateral ventricle and microvessels of adjacent brain.

The distribution of gamma-glutamyl transpeptidase in different vascular compartments of the central nervous system was evaluated in several common laboratory animals, i.e., hamster, gerbil, guinea pig, rat and mouse, by enzyme-histochemistry. Microvascular endothelium of the periventricular brain tissue stained positively in all five species. In contrast, the vascular endothelium of the choroid plexus stained positively only in the gerbil, and was negative in the other four species. Positive reactions for the transpeptidase was also found in choroid plexus epithelial cells in guinea pig, rat, and mouse; however no activity could be demonstrated in these cells of hamster and gerbil. The results demonstrate clear species differences in localization of the enzyme and suggest that gamma-glutamyl transpeptidase-promoted amino acid transport in choroid plexus is different in various animal species. It is also suggested that in gerbil, transpeptidase-aided amino acid transport takes place in endothelial cells of choroid plexus, whereas in guinea pig, rat and mouse this occurs in epithelial cells of choroid plexus. In the case of hamster, such aided transport is absent in endothelial as well as in epithelial cells of the choroid plexus. Thus, the hamster and the gerbil showed differences in gamma-glutamyl transpeptidase distribution, whereas the guinea pig, rat, and mouse showed similar enzyme distributions.

Membrane-bound proteases of the gerbil subfornical organ and choroid plexus: an enzyme histochemical study.

Using enzyme-histochemical methods, the membrane-bound peptidases, gamma-glutamyl transpeptidase (gamma-GTP), microsomal alanyl aminopeptidase (mAAP), glutamyl aminopeptidase (EAP), and dipeptidyl peptidase IV (DPP IV), were studied in microvessels of the gerbil subfornical organ (SFO), choroid plexus adjacent to the SFO, and the ependyma of brain ventricle walls in the vicinity of the SFO. Vessels and microvessels of gerbil SFO and choroid plexus were positive for gamma-GTP, mAAP, and EAP, but negative for DPP IV. Blood-brain barrier (BBB) microvessels in the surrounding brain tissue also showed positive reactions for gamma-GTP, mAAP, and EAP but a negative reaction for DPP IV. Both epithelial cells of the choroid plexus and ependymal cells of the ventricle walls were negative for all four studied enzymes. It is suggested that blood-borne peptide hormones which can be substrates for these membrane-bound proteases can be modulated by gamma-GTP, mAAP, and EAP, but not by DPP IV, when they come in contact with the plasma membrane of the endothelial cells of the vessels in gerbil SFO, choroid plexus, and surrounding brain tissue.

Sequential renal alterations in septic shock in the primate.

This is a descriptive sequential study of the response of the baboon to LD100 Escherichia coli. The response was found to consist of three stages based on electron microscopic, physiologic, and clinical laboratory data. This study associates the inflammatory, coagulant, and cell injury (stage 1-3) responses with markers of activation of inflammatory cells (tumor necrosis factor) and of the vascular endothelium (tissue plasminogen activator). This work also shows that in contrast to the underlying parenchymal cells of the organ, the vascular endothelium remains intact throughout the response to LD100 E. coli. The possible role of the vascular endothelium in mediation of events at both its luminal (blood) and antiluminal (parenchymal) surfaces is discussed.

Distribution of human leukocyte antigen-ABC and -D/DR antigens in the unfixed human testis.

The distribution of the major histocompatibility complex (MHC) antigens in the unfixed human testicle was studied by indirect immunofluorescence. Three murine monoclonal antibodies to the common determinants of class I MHC antigens (human leukocyte antigen [HLA]-ABC) and three against class II MHC antigens (HLA-D/DR antigens), respectively, were utilized. No class I MHC antigens were identified on developing testicular germ cells including spermatozoa, but interstitial cells between the seminiferous tubules (including Leydig cells) and blood vessel endothelium expressed the antigen. Class II MHC antigens were not found on any cells within the seminiferous tubules. However, the class II antigen was identified on dendritic-like cells between the seminiferous tubules and on vessel endothelium, although its expression was expectedly limited. These findings indicate that human testicular germ cells express minimal or no MHC antigens.

Preliminary report: immunoperoxidase-linked human placental lactogen as a histopathologic index of perinatal morbidity and mortality.

Anatomic study of placental dysfunction may benefit from the applications of new routine techniques. Immunohistochemical staining for human placental lactogen (HPL) was used in 3 cases illustrating different perinatal disorders. The amount of HPL labeling ranged from high in an acute anoxic death due to abruptio placentae, through decreased in a case of maternal hypertension, to low in severe intrauterine growth retardation. Such information complements standard clinical and pathologic studies. Ten percent buffered Formalin was superior to Bouin's fixative and alcoholic Formalin for the demonstration of HPL. Even after 4 days of refrigeration at 4 degrees C, all of the syncytiotrophoblastic tissue was labeled in sections of paraffin-embedded, Formalin-fixed normal placentas.

Cycling of peripheral blood and marrow lymphocytes in cyclic neutropenia.

A 16-month-old male patient with cyclic neutropenia was found to have cyclic fluctuations of monocytes, lymphocytes, platelets, and eosinophils in the peripheral blood. Changes in lymphocyte counts were not obviously related to B, T, or natural killer cells. All classes of immunoglobulins were elevated throughout the cycle. Studies of the marrow morphology revealed remarkable cyclic oscillations of lymphoid as well as myeloid lineage cells. Granulocyte-macrophage progenitors (CFU-c) cycled and were virtually absent 1 wk prior to the neutropenic nadir. The cyclic changes in marrow lymphoid cell numbers were primarily due to changes in numbers of surface immunoglobulin negative (sIg-), cytoplasmic Ig+ (cIg+) pre-B cells. Pre-B cell numbers cycled from normal to extraordinarily elevated values with the same periodicity but reciprocal to the neutrophil cycle. We propose that the primary defect in cyclic neutropenia may either be a periodic failure of an early myeloid differentiation factor or a blunted response of early myeloid precursors to a common hemopoietic growth factor. This may lead to periodic fluctuations in the production or delivery of growth factors (or factor) that influence early stages of differentiation of other hemopoietic cells, including pre-B cells. The essential periodic deficiency is consequently reflected in deficient production of CFU-c accompanied by excessive production or accumulation of pre-B cells (and probably other hemopoietic precursors) in the marrow.

Macromolecular syntheses related to the reproductive cyst of Tetrahymena patula.

Macromolecular syntheses in encysted Tetrahymena patula were studied using Feulgen fluorescence cytophotometry, autoradiography, and inhibitors of RNA and protein synthesis. Cycloheximide significantly depressed protein synthesis and D-actinomycin effectively blocked RNA synthesis. Under these conditions, the cells within the cyst were unable to divide. Both cytophotometric measurements and autoradiographic data with tritiated thymidine show that DNA synthesis does not occur during the encystment divisions. Excysted cells placed in nutrient broth medium showed a prolonged generation time after the first cell growth cycle, and by the third generation the mean DNA content per cell was almost triple that of starved excysted cells. These findings indicate that (a) the encystment divisions require RNA and protein synthesis, which are apparently effected through turnover, (b) the encystment division cycles occur in the absence of DNA synthesis, and (c) excysted cells placed in culture medium may go through more than one DNA replication per cell cycle.