A Highly Sensitive Radioimmunoassay for Human Growth Hormone Using a Monoclonal Antibody

Biozzi-strain mice were immunized with a highly purified preparation of 20K wriant of hGH. Spleen-cells were fused with SP2/0Ag14 myeloma cells. Clone productions were screened for specificity toward 20K and 22K hGH and for the affinity constant of antibody-antigen reaction. For the selected monoclonal antibody, Ka was 1.02.10¹¹ L/M using 22K hGH as both tracer and reference preparation. No cross reactivity was found with PRL and other pituitary hormones; hPL reactivity was 0.002 percent that of hGH. According to these antibody characteristics, a highly sensitive RIA system was developed and used for specific GH measurement in human serum. Using logit-log co-ordinates, the slope of the standard curve was -1.099 and the minimum detected dose was 0.5 uIU/ml.

Excellent correlation (r=0.9575) was found between assay data in this system and those of a conventional RIA method using specific polyclonal rabbit antiserum.

The International Reference preparation (66/217) could adequately be used to calibrate the monoclonal antibody system since the in house internal 22K GH standard and international one were equally well recognized by the monoclonal antibody.     

CD8+ T-cell recognition of a synthetic epitope formed by t-butyl modification

We set out to clone Bax-specific CD8⁺ T cells from peripheral blood samples of patients with primary chronic lymphocytic leukaemia. A number of clones were generated using a Bax peptide pool and their T-cell epitope was mapped to two peptides sharing a common 9-amino-acid sequence (LLSYFGTPT), restricted by HLA-A*0201. However, when these T-cell clones were tested against highly purified syntheses (> 95%) of the same peptide sequence, there was no functional response. Subsequent mass spectrometric analysis and HPLC fractionation suggested that the active component in the original crude peptide preparations (77% pure) was a peptide with a tert-butyl (tBu) modification of the tyrosine residue. This was confirmed by modification of the inactive wild-type sequence to generate functionally active peptides. Computer modelling of peptide:HLA-A*0201 structures predicted that the tBu modification would not affect interactions between peptide residues and the HLA binding site. However, these models did predict that the tBu modification of tyrosine would result in an extension of the side chain out of the peptide-binding groove up towards the T-cell receptor. This modified product formed < 1% of the original P603 crude peptide preparation and < 0·05% of the original 23-peptide mixture used for T-cell stimulation. The data presented here, illustrate the potential for chemical modifications to change the immunogenicity of synthetic peptides, and highlight the exquisite capacity of T-cell receptors to discriminate between structurally similar peptide sequences. Furthermore, this study highlights potential pitfalls associated with the use of synthetic peptides for the monitoring and modulating of human immune responses.     

Receptors for neurohypophyseal hormones along the rat nephron: 125I-labelled d(CH2)5[Tyr(Me)2, Thr4, Orn8, Tyr-NH 2 9 ] vasotocin binding in microdissected tubules

A microassay was developed to measure the binding of the labelled monoiodinated analogue [1-(mercapto-,-cyclopentamethylenepropionic acid), 2-O-mithyltyrosine, 4-threonine, 8-ornithine, 9-¹²⁵I-tyrosylamide]vasotocin ¹²⁵I-d(CH₂)₅[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT to isolated nephron segments microdissected from collagenase-treated rat kidneys. When determined using 1.7 nM labelled ligand at 4° C, specific binding sites (expressed at 10^–18 mol ¹²⁵I-d(CH₂)₅[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT bound/mm tubule length) were found in medullary thick ascending limbs (MTAL), 1.67±0.49; cortical thick ascending limbs, 2.20±0.80; cortical collecting ducts, 2.39±0.86; outer medullary collecting ducts (OMCD), 2.54±0.53 and inner medullary collecting ducts, 5.33±0.40, whereas no specific binding could be detected in glomeruli and proximal tubules. Specific ¹²⁵I-d(CH₂)₅[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT binding to OMCD was saturable with incubation time and reversible after elimination of free labelled ligand (the association and dissociation rate constants at 4° C were 1.06×10⁷ M^–1 min^–1 and 1.95×10^–2 min^–1 respectively). The stereospecificity of MTAL and OMCD binding sites was assessed in competitive experiments revealing the following recognition pattern for a series of eight vasopressin analogues:ddAVP>AVP>d(CH₂)₅-[Tyr (Me)², Thr⁴, Tyr-NH ₂ ⁹ ]OVT=AVT=OT>d(CH₂)₅[Tyr(Me)²]AVP=[Thr⁴, Gly⁷]OT>[Phe², Orn⁸]VT, whereas pharmacological concentrations of insulin and glucagon did not impair radioligand binding. These results indicate that the detected labelled binding sites might correspond mainly to physiological V₂ vasopressin receptors.     

Stimulatory actions of di-8-butyl-amino-naphthyl-ethylene-pyridinium-propyl-sulfonate (di-8-ANEPPS), voltage-sensitive dye, on the BKCa channel in pituitary tumor (GH3) cells

Di-8-ANEPPS (4-{2-[6-(dibutylamino)-2-naphthalenyl]-ethenyl}-1-(3-sulfopropyl)pyridinium inner salt) has been used as a fast-response voltage-sensitive styrylpyridinium probe. However, little is known regarding the mechanism of di-8-ANEPPS actions on ion currents. In this study, the effects of this dye on ion currents were investigated in pituitary GH₃ cells. In whole-cell configuration, di-8-ANEPPS (10 μM) reversibly increased the amplitude of Ca²⁺-activated K⁺ current. In inside-out configuration, di-8-ANEPPS (10 μM) applied to the intracellular surface of the membrane caused no change in single-channel conductance; however, it did enhance the activity of large-conductance Ca²⁺-activated K⁺ (BK_Ca) channels with an EC₅₀ value of 7.5 μM. This compound caused a left shift in the activation curve of BK_Ca channels with no change in the gating charge of these channels. A decrease in mean closed time of the channels was seen in the presence of this dye. In the cell-attached mode, di-8-ANEPPS applied on the extracellular side of the membrane also activated BK_Ca channels. However, neither voltage-gated K⁺ nor ether-à-go-go-related gene (erg)-mediated K⁺ currents in GH₃ cells were affected by di-8-APPNES. Under current-clamp configuration, di-8-ANEPPS (10 μM) decreased the firing of action potentials in GH₃ cells. In pancreatic βTC-6 cells, di-8-APPNES (10 μM) also increased BK_Ca-channel activity. Taken together, this study suggests that during the exposure to di-8-ANEPPS, the stimulatory effects on BK_Ca channels could be one of potential mechanisms through which it may affect cell excitability.     

Enamel Specific Protein Kinases and State of Phosphorylation of Purified Amelogenins

Ameloblastic tissue samples from unerupted bone molars were used to prepare subcellular enamel protein kinase preparations, nuclear+plasma membrane, cytosolic and microsomal, and used in in vitro phosphorylation of purified 20 kDa bovine amelogenin in the presence of ²P-ATP. Both cytosolic and microsomal preparations can phosphorylate purified native amelogenins, the addition of Ca²⁺ slightly increased the microsomal enzyme activity or at least did not inhibit the activity, whereas the presence of Ca²⁺ substantially decreased the cytosolic kinase activity towards phosphorylation of amelogenins. A comparative analysis using the enamel microsomal kinase against osteopontin, dephosphorylated casein and bone sialoprotein showed no phosphorylation of the first two proteins, and only minor phosphorylation of the bone sialoprotein. Overall, the present work demonstrates for the first time that the protein kinase responsible for the phosphorylation of amelogenins is a novel kinase, which is not inhibited by Ca²⁺, unlike the microsomal protein kinase (casein kinase type-II) of bone which phosphorylates secretory proteins osteopontin and bone sialoprotein and is strongly Ca²⁺ inhibited. The direct phosphoserine analysis on the purified bovine 20 kDa amelogenin indicated the presence of 0.8 moles of phosphoserine/mole protein naturally occurring, consistent with the quantitative analysis of ¹⁴C-radiolabeling of phosphoserines by conversion to dehydroalanine and in situ reaction with the thiol agent, ¹⁴C-mercaptoethanol, 0.64 moles ^uC-incorporated/mole 20 kDa amelogenin. The purified low Mramelogenins 5.3 kDa E4 (TRAP) and 7.2 kDa E3 (LRAP), were also derivatized by ¹⁴C-mercaptoethanol, providing 0.46 and 0.88 moles ¹⁴C-incorporated/mole respectively. Further studies of the ¹⁴C-radiolabeled E4 amelogenin by sequence analysis confirmed one site of label to be at position 16 from the N-terminal and hence provided a direct evidence for the naturally occurring phosphoserine residue at this position.     

Effects of CO2, acetylcholine and caerulein on45Ca efflux from isolated mouse pancreatic fragments

Mouse pancreatic fragments were loaded with⁴⁵Ca and placed in a flow cell. The concentration of⁴⁵Ca in the effluent was measured. The effects of changing the tension of carbon dioxide on⁴⁵Ca efflux were observed and compared with effects of pancreatic secretagogues.The normal control solution was equilibrated with 5% CO₂, 95% O₂. Shift to solutions equilibrated with 10, 20, 50 or 100% CO₂ evoked a dose-dependent increase in fractional⁴⁵Ca efflux, with a just detectable effect at 10% and a maximal one at 50%.The CO₂-evoked Ca release was not due to anoxia, since a short period of exposure to a 100% N₂-equilibrated solution had no effect. A decrease in extracellular pH (tris buffering) had only a very modest effect on⁴⁵Ca efflux.CO₂-evoked Ca release under conditions avoiding extracellular pH changes (20% CO₂, 100 mM NaHCO₃). This CO₂-evoked enhanced⁴⁵Ca efflux was sustained during a 30 min stimulation period, but was abruptly terminated on return to the control solution (5% CO₂, 25 mM NaHCO₃). NH₃ (10 mM) added to the 20% CO₂-equilibrated solution for a brief interval in the middle of a period of CO₂-evoked enhanced⁴⁵Ca efflux evoked a rapid return of the fractional Ca efflux towards the resting level. This effect was rapidly reversible.While the CO₂-evoked Ca release was largely sustained, the ACh-evoked increase in⁴⁵Ca fractional efflux was entirely transient. The CO₂-evoked Ca release was not inhibited by a background of sustained ACh stimulation. ACh-evoked Ca release, however, was markedly inhibited in the presence of sustained CO₂ stimulation.2,4 Dinitrophenol (1 mM) in combination with iodoacetate (2 mM), while markedly reducing⁴⁵Ca uptake into the fragments during the loading period had little or no effect on the ACh-evoked increase in⁴⁵Ca fractional efflux. The CO₂-evoked Ca release, however, was markedly reduced by these metabolic inhibitors.The local anaesthetic procaine (1 mM) virtually abolished ACh- or caerulein-evoked Ca release without having any influence on the CO₂ effect.It is concluded that CO₂ releases Ca from pancreatic acinar cells by means of intracellular acidification. This effect may in part be due to H⁺ displacement of Ca²⁺ from intracellular membrane binding sites and partly due to release of Ca from compartments (organelles) into which Ca has been actively accumulated.     

Intrarenal distribution of125I-polyvinyl pyrrolidone and51Cr-labelled red cells in hydropenic and polyuric rabbits

Summary The intrarenal volumes of distribution of¹²⁵I-polyvinyl pyrrolidone and⁵¹Cr-labelled red cells have been examined in rabbits under conditions of severe hydropenia and starvation-induced polyuria, and in rabbits under control conditions. The PVP space was significantly reduced, and the ratio [red cell space]:[red cell space + PVP space] significantly elevated, in the outer cortex of kidneys of hydropenic rabbits, and in the papilla of polyuric rabbits. It is suggested that the former may in some manner result from a reduction in the total cortical vascular space, whereas the latter reflects variations of medullary and appillary interstitial tonicity under these conditions.     

Overexpression of CD39 and high tumoral CD39+/CD8+ ratio are associated with adverse prognosis in resectable gastric cancer

CD39/ectonucleoside triphosphate diphosphohydrolase-1 (ENTPD1) is a cell surface-located, rate-limiting enzyme in the generation of adenosine, and plays a crucial role in tumor development. We examined co-expression of CD39 and CD8in gastric cancer (GC) and showed that the expression of CD39 and CD8 increased significantly in tumor tissues compared to paired peritumor tissues. The expression of tumoral CD39 (tCD39), but not tumoral CD8 (tCD8), was related to overall survival. Furthermore, the CD39⁺/CD8⁺ ratio was associated with poor prognosis in resected GC patients. Taken together, our data indicate that highCD39 expression and high tCD39⁺/CD8⁺ ratio in GC is a predictor of poor prognosis for GC patients after radical resection. Moreover, CD39 could serve as a potential target for cancer immunotherapy.     

Cyclophosphamide inhibits the generation and function of CD8(+) regulatory T cells

CD8(+) regulatory T cells (Treg) and CD4(+)CD25(+) Treg infiltrate human cancers, thus favoring tumor immune escape. Therefore, in the setting of antitumor therapeutic protocols, it is important to associate antitumor treatment with agents that are able to inhibit Treg function. Cyclophosphamide (CY) has been demonstrated to be effective in counteracting CD4(+)CD25(+) Treg activity. Hence, we tested its inhibitory efficacy on human CD8(+) Treg. Because CY is a prodrug, 4-hydroperoxycyclophosphamide (4-HC), a derivative of CY that in aqueous solution is converted to 4-hydroxycyclophosphamide, an active metabolite of CY, was used. 4-HC significantly inhibited CD8(+) Treg generation and function but only at the higher tested concentration (0.5 μg/mL), that is, in the therapeutic range of the drug. The lower 4-HC concentration tested (0.1 μg/mL) was almost ineffective. 4-HC inhibitory effects were related to apoptosis/necrosis induction. When CD8(+)CD28(+) non-Treg were analyzed for comparative purposes, significantly lower cytotoxic rates among these cells were observed than among CD8(+) Treg, which were differentiated because they did not express the CD28 molecule. These data demonstrate that CD8(+) Treg are inhibited through cytotoxic phenomena by CY, thus supporting the use of this drug at adequate concentrations and schedules of administration as a Treg inhibitor in combinatorial chemo- or immunotherapeutic anticancer protocols.     

Topography of 3H-DHA, 3H-QNB, 3H-Dopamine, and 3H-DAGO Binding Sites Distribution in the Central Part of the Sinoatrial Node in Rat Heart

The topography of distribution of ³H-dihydroalprenolol, ³H-quinucledinyl benzilate, ³H-dopamine, and ³H-DAGO binding sites in the central part of the sinoatrial node in rat heart was studied by autoradiography after electrophysiological identification of the dominant pacemaker region location. Receptor asymmetry between the lateral and median regions of the central part of the sinoatrial node was shown. The dominant pacemaker region lay in the lateral area of the sinoatrial node; the number of binding sites for all four ligands was minimum in it. The number of binding sites gradually increased in the cranial and caudal directions from the dominant pacemaker region along the sinoatrial node artery (more smoothly in the caudal direction). The relative densities of bindings sites for ³H-dihydroalprenolol and ³H-dopamine were higher in the lateral region compared to the perinodal working myocardium, while the densities for ³H-quinucledinyl benzilate and ³H-DAGO were virtually the same. The distribution of binding sites along the artery in the median region of the sinoatrial node was even for ³H-quinucledinyl benzilate and ³H-DAGO. For ³H-DAGO these parameters were close to those in the perinodal atrial myocardium, for ³H-quinucledinyl benzilate somewhat lower. Curves presenting the distribution of binding site densities for ³H-dihydroalprenolol and ³H-dopamine in the median region of the sinoatrial node were similar, with a pronounced peak in the region contralateral to the dominant pacemaker region, and significantly higher binding parameters compared to those for the perinodal atrial myocardium. The difference consisted in higher density of ³H-dopamine binding sites in the median region of the sinoatrial node in comparison with the lateral region. Binding activity was maximum in the wall of the sinoatrial node artery. The distribution of binding sites for ligands to the main autonomic nervous system neurotransmitters in the rat heart sinoatrial node is heterogeneous. __________ Translated from Byulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 140, No. 10, pp. 472–477, October, 2005     

CD4(+) and CD8(+) cells are key participants in the development of recurrent herpetic stromal keratitis in mice

Ocular herpes simplex virus (HSV) infection results in an immune-mediated inflammation of the corneal stroma known as herpetic stromal keratitis (HSK). Recurrent HSK is a common cause of virus-induced corneal blindness in humans. The role of CD4(+) and CD8(+) T cell subsets in the disease pathogenesis is ill defined and varies with the virus strain and host genetic background. To examine the contribution of T cell subsets to corneal disease, we studied the development of recurrent HSK in CD4 or CD8 gene knockout (KO) mice ocularly infected with HSV-1 McKrae strain. Following UV-B induced viral reactivation, corneal opacity in latently infected BALB/c (HSV sensitive) CD4 and CD8 KO mice was reduced compared to infected BALB/c mice with normal genotype. In contrast, opacity in C57BL/6 (HSV resistant) CD4 and CD8 KO latent mice did not differ from genetically normal latent mice. Virus-induced corneal opacity was not demonstrable in C57BL/6 CD4/CD8 double KO mice. Increased viral shedding, measured by reactivation rate, days shedding or viral titers, occurred in CD4 KO mice of both strains. Our findings indicate that both CD4(+) and CD8(+) cells play a role in the immunopathogenesis of recurrent HSK, and their role is dependent upon the host genetic profile.     

Antigen-nonspecific activation of CD8+ T lymphocytes by cytokines: relevance to immunity, autoimmunity, and cancer

Development of T lymphocytes and their survival in the periphery are dependent on signals emanating from cytokine receptors as well as the T cell antigen receptor (TCR). These two signaling pathways play distinct and complementary roles at various stages of T cell development, maturation, survival, activation and differentiation. During immune response to foreign antigens initiated by TCR signaling, cytokines play a key role in the expansion of activated T cells. Even though the initial activation of T cells occurs via the TCR, this requirement can be overcome under certain circumstances. During lymphopenia, cytokines trigger memory CD8⁺ T cells to undergo antigen non-specific homeostatic expansion, whereas naïve CD8⁺ T cells require both cytokines and TCR signaling. Recent reports show certain combinations of cytokines can induce proliferation and effector functions of naïve CD8⁺ T cells without concomitant stimulation via the TCR. While such antigen non-specific stimulation of naïve T cells might significantly boost the adaptive immune response, it could also have an undesirable effect of triggering potentially autoreactive cells. Understanding the mechanisms and the regulation of cytokine-driven stimulation of naïve CD8⁺ T cells may lead to novel strategies of intervention for autoimmune diseases. On the other hand, in vitro expansion of naïve CD8⁺ T cells by certain combinations of cytokines could be used to generate tumor-specific cells with ideal properties for cellular immunotherapy of cancer.     

An efficient and simple method of non-enzymatic detachment of monolayer cultures into single cell suspension

Summary Activating the Na⁺/H⁺ antiporter causes a distinctive and rapid endocytosis which internalizes the plasma membrane leading to cell retraction and detachment as rounded cells with much reduced surface area. Antiport activation is by a combination of two individually effective motivations, viz. a) steep [Na⁺] and [H⁺] gradients across the plasma membrane and b) allosteric activation by second messengers initiated with simple inorganic sulphate. The dissociated cells are as viable as those released by trypsinization. Thus it provides an effective enzyme-free alternative to trypsin digestion in cell dissociation.     

Functional Modifications of Tee Cd4+and the Cd8+-H Subset Due to Maternal Serum

Abstract

Numerous experiments have been performed to try to explain the successful gestation of the semiallogeneic mammalian fetus in the immuno-competent mother (1, 3, 4, 5). A popular hypothesis is that localized intra uterine suppression mediates the immune response and contributes directly to the survival of the fetus (6). Suppressive factors synthesized at the fetomaternal interface may be transported by the bloodstream and be found in the retroplacental and peripheral blood circulation (7). In this report we aim to study these modulating factors by exposing a proteinaceous antigen (Candidine or human mono nuclear cells) to unrelated human lymphocytes in the presence of a pool of maternal serum, retroplacental (MAT SR) or peripheral (MAT SP), or to a pool of male or calf serum (MALS or CS).

A significant downregulation of the candidine mediated lymphocytic stimulation was observed in the case of the maternal serum. In order to further characterize this inhibition, a quantitative evaluation of the expression of the CD₄ and CD₈ positive subpopulations was performed. A selective inhibition of the CD₄ positive subset was observed.

In the PHA stimulation assay in the presence of maternal serum there was an inhibition of the thymidine uptake of unrelated lymphocytes. When studying the different subsets which were stimulated in the presence of maternal serum and control media it was shown that the CD₄ positive subpopulation remained unchanged while there was a slight inhibition of the CD₈ positive subset in the first case (maternal serum treated).

The same CD₄ positive inhibitive property of maternal serum was observed when a neoplastic cell line (HUT cells) was used as target for candidine in a stimulation test. This CD₄ inhibited expression remained constant as long as the maternal serum was renewed.

Maternal lymphocytes however remained resistant to the inhibitive action of maternal serum and did not show any change in their CD₄ and CD₈ positive subpopulation. By investigating the different blood components, it was shown that the suppressive factor was included in the IgG fraction and synthesized at the placental level. Appearing quite early during the gestation (at the 5-6th week), this factor was vanishing 2 weeks after the delivery.     

Electron microscopic radioautographic study on the syntheses of DNA, RNA and protein in the livers of aging mice

The syntheses of DNA, RNA and protein in the livers of aging mice was studied by electron microscopic radioautography using radioactive precursors. Labeling with³H-thymidine was observed in the nuclei of some hepatocytes at various prenatal and postnatal ages. The percentage of labeled cells decreased after birth, then slowly fell to the lowest value at 2 years. The silver grains with³H-uridine labeling were observed in both the nuclei and cytoplasm of hepatocytes at various ages. There was a peak in uridine labeling at 14 days, and then it slowly decreased until old age. The number of silver grains with³H-leucine labeling in the cytoplasm of hepatocytes was small. It increased after birth, reached the maximum at 1 month, and continued to decrease with aging.     

A radioimmunoassay for the determination of insulins from several animal species, insulin derivatives and insulin precursors in both their native and denatured state

Antibodies were raised against the carboxy terminus of the insulin A chain in sheep, goat and rabbit using as antigen the synthetic octapeptide YQLENYCN conjugated to bovine serum albumin (BSA). All of the antisera obtained cross-reacted with molecules containing this peptide sequence. Using these antibodies, we developed a radioimmunoassay that could detect the insulin A chain itself, both denatured and natural mammalian and avian insulins, as well as proinsulins and insulin fusion proteins from microorganisms.     

Analysis of Human Immunodeficiency Virus Type 1 Integration by Using A Specific, Sensitive and Quantitative Assay Based on Real-time Polymerase Chain Reaction

A novel real-time nested-PCR assay was developed to quantify integrated human immunodeficiency virus type-1 (HIV-1) DNA with high specificity and sensitivity. This assay reproducibly allowed the detection of three copies of integrated HIV DNA in a background of 100,000 cell equivalents of human chromosomal DNA. The non-specific amplification of unintegrated HIV-1 DNA was significantly inhibited in this assay and the specificity of this assay was much higher than the previously reported method. This assay showed that kinetics in viral DNA sysnthesis was cell-type dependent and that the kinetics of HIV-1 DNA integration was very rapid in Jurkat T cell line. This method may provide new insights into the integration processes and be useful in evaluating future integrase inhibitors.     

Morphological demonstration and quantification of TSH binding sites in neoplastic and non-neoplastic thyroid tissues

Summary We have developed a morphological method to portray TSH binding sites in intact tissue specimens. Frozen sections were incubated with¹²⁵I-labelled TSH so as to localise binding sites by autoradiography. The proof of specificity was substantiated by: the competitive inhibition of¹²⁵I-TSH-labelling with cold TSH, the lack of binding in non-target tissues and a lack of binding in TSH target tissues after incubation with¹²⁵I-hCG or free¹²⁵I.In applying this method to a total of 22 surgical specimens of thyroid, striking differences came to light in respect of the degree to which¹²⁵I-TSH binding occurred in the various thyroid disorders. When compared with histologically normal tissue, labelling was generally decreased in toxic adenomas, non-functioning adenomas (cold nodules), and thyroids affected by Graves' disease, whereas non-toxic colloid goitre cases clearly exhibited denser binding. Medullary and anaplastic carcinomas exhibited no specific labelling whilst binding varied in the differentiated carcinomas between no effective binding or a level resembling that found in normal thyroid tissue.     

Electrophysiological analysis of rat renal sugar and amino acid transport

Transepithelial and cellular electrical potential changes were measured in response to luminal perfusion ofd-glucose and related substrates in micropuncture experiments on rat kidney in vivo. By studying the dependence of the potential response on various experimental parameters, some insight was obtained into the mechanism of Na⁺ coupled glucose absorption. The experiments confirm the driving forces for glucose absorption in the living cell to be: a) the Na concentration gradient, b) the electrical potential gradient and c) the glucose concentration gradient across the brushborder membrane. Furthermore they describe the substrate specificity of the cotransport mechanism and the mechanism of inhibition ofd-glucose transport by various inhibitors, such as phlorizin, harmaline and oubain. The latter experiments suggest that the active Na⁺ pump in the peritubular cell membrane, which establishes the Na⁺ ion gradient and the electrical potential gradient across the brushborder, contributes a measurable partial conductance to the overall electrical conductance of the peritubular cell membrane.     

Relative importance of CD4+ and CD8+ T cells in the resolution of Chlamydophila abortus primary infection in mice

The role of the specific cellular immune response is well established in Chlamydiaceae infections, but the importance of each T-cell subset seems to be species-dependent. This study was designed to clarify the role of T-cell subsets in the response to Chlamydophila abortus primary infection. C57BL/6 mice were depleted of CD4+ or CD8+, or both, by monoclonal antibody injections and subsequently infected with C. abortus. Mice were killed at intervals and samples were collected for bacteriological and histopathological analysis. Also carried out were spleen cell culture, cytokine quantification, immunolabelling for C. abortus antigen, and a TUNEL assay for apoptosis. CD8+ T cell-depleted mice all died within 12 days of C. abortus infection, while no mortality was observed in the other groups; surprisingly, CD4+ T cell-depleted mice showed lower morbidity (expressed as weight loss) than did a non-depleted (control) group. CD8+ T cell-depleted mice also differed from the other groups in showing a significantly higher chlamydial burden in the liver. CD8+ T cell-depleted mice also had a higher number of apoptotic cells in hepatic inflammatory foci and showed exacerbated IFN-gamma production by spleen cells after specific stimulation. Simultaneous depletion of both T-cell subpopulations led to a chronic infection, but not to early mortality. It is concluded that CD8+ T cells may play a role in the regulatory control of the CD4+ T-cell response and may have a direct cytotoxic or IFN-gamma-mediated effect on infected cells.