Long-term trends in HIV care entry: over 15 years of clinical experience from Poland.

INTRODUCTION: Delay in HIV diagnosis and consequently late care entry with low CD4 counts remain a major challenge for the control of the HIV/AIDS epidemic. The aim of this study was to analyse the evolution of characteristics of the HIV epidemic in Poland. METHODS: Cross-sectional data were collected for 3972 HIV-infected patients followed up in 14 of 17 Polish HIV treatment centres in the years 2000-2015. Clinical data were analysed and factors associated with late presentation (baseline CD4 count < 350 cells/µL or history of AIDS-defining illness) and advanced HIV disease (baseline CD4 count < 200 cells/µL or history of AIDS) were identified. RESULTS: The majority (57.6%) of patients entered care late, while 35.6% presented with advanced HIV disease. The odds of being linked to care late or with advanced HIV disease increased consistently across age categories, increasing from 2.55 [95% confidence interval (CI) 1.46-4.47] for late presentation and 3.13 (95% CI 1.49-6.58) for advanced disease for the 21-30-year-old category to 5.2 (95% CI 1.94-14.04) and 8.15 (95% CI 2.88-23.01), respectively, for individuals > 60 years of age. Increased risks of late entry and advanced HIV disease were also observed for injecting drug users [adjusted odds ratio (aOR) 1.74 (95% CI 1.16-2.60) and 1.55 (95% CI 1.05-2.30), respectively], with lower aOR associated with the men who have sex with men transmission route [aOR 0.3 (95% CI 0.31-0.59) and 0.39 (95% CI 0.29-0.53), respectively]. The frequencies of cases in which patients were linked to care late and with advanced HIV disease decreased over time from 67.6% (2000) to 53.5% (2015) (P < 0.0001) and from 43.5% (2000) to 28.4% (2015) (P = 0.001), respectively. CONCLUSIONS: Despite improvements over time, most patients diagnosed with HIV infection entered care late, with a third presenting with advanced HIV disease. Late care entry remains common among people who inject drugs and heterosexual groups.

Muscarinic cholinergic receptors of rat lymphocytes: effect of antigen stimulation and local brain lesion.

Expression of muscarinic cholinergic receptors (m-AchR) on thymocytes and lymphocytes supports a hypothesis of direct interaction between autonomic innervation and the immune system. Using a muscarinic antagonist, [3H]quinuclidinyl benzilate ([3H]QNB), we revealed different characteristics of m-AchR expression in lymphocytes isolated from the spleen, peripheral blood or thymus of Wistar rats. To further explore the mechanisms controlling m-AchR expression in lymphocytes we have addressed two questions: (i) whether local brain lesions will affect [3H]QNB binding activity of lymphocytes and (ii) whether different antigens will alter m-AchR expression in lymphocytes. We report here that electrolytic lesions of the area hypothalamica anterior resulted in a significant increase in [3H]QNB-specific binding in thymocytes 7 days after neurosurgery. In contrast, local lesions of other hypothalamic structures (area preoptica medialis, area hypothalamica posterior) or sensomotor cortex did not affect m-AchR expression in thymocytes. No apparent changes of [3H]QNB-binding activity were found in lymphocytes isolated from rat spleen or peripheral blood after operation. Both T-cell-dependent (sheep red blood cells) and T-cell-independent (Vi antigen of Salmonella typhi) antigens induced a significant increase in [3H]QNB-specific binding in spleen lymphocytes while [3H]QNB-binding activity of peripheral blood lymphocytes did not change. A different pattern of lymphocyte m-AchR expression found in various lymphoid tissues after immunization or brain lesions suggests a local involvement of cholinergic mechanisms in neuroimmune interaction.

Muscarinic antagonist binding to intact rat thymocytes.

The presence and properties of muscarinic receptors on intact rat thymocytes have been studied by the use of the muscarinic antagonists [3H]-3-quinuclidinyl benzilate (3H-QNB), [3H]-4-N-methylpiperidinyl benzilate ([3H]-NMBP) and [3H]-N-methylscopolamine (3H-NMS). The course of binding of 3H-QNB reveals a maximum at 5 min, and shows a subsequent decrease of bound radioactivity, suggesting internalization of the receptor 3H-3-QNB complex. This phenomenon has also been studied by the use of another muscarinic antagonist, 3H-NMPB, which has a faster on and off rate than 3H-QNB, and which may be rapidly displaced by an excess of unlabeled muscarinic antagonist, atropine. Bound 3H-NMPB is highly susceptible to atropine displacement only within the first two minutes of incubation with thymocytes at 37 degrees C. The kinetics of binding of the less lipophilic muscarinic antagonist 3H-NMS to thymocytes, show no maximum as a function of incubation time and 3H-NMS is susceptible to displacement by atropine up to 30 min incubation time with the thymocytes. These data suggest that binding of lipophilic benzilate type antagonists (3H-QNB and 3H-NMPB) may be followed by internalization and/or isomerization of the receptor-benzilate antagnist complex while the 3H-NMS-receptor complex is not subject to these processes.

Cholinergic muscarinic and nicotinic binding by human lymphocytes: differences between peripheral blood cells and cultivated cell lines.

Kinetic and equilibrium studies of [3H]QNB and [3H]nicotine binding to human peripheral blood lymphocytes have been performed. The calculated number of muscarinic binding sites is 6 x 10(4) per cell, and of nicotinic binding sites is 2 x 10(3) per cell. The conditions for routine estimations of muscarinic and nicotinic receptors on human lymphocytes have been established. Significant differences in the distribution of muscarinic and nicotinic binding sites among normal PBL and malignant cell lines have been described.

Expression of muscarinic cholinergic receptors during T cell maturation in the thymus.

Thymocytes at various stages of their ontogeny have been studied in relation to their ability to bind [3H]quinuclidinyl benzilate [( 3H]QNB), a specific radioligand of the muscarinic cholinergic receptors. [3H]QNB-specific binding to thymocytes from 15-19-day fetal, newborn and adult thymuses of mice and rats was compared and correlated. Our experiments showed that the kinetics of [3H]QNB binding to thymocytes at 37 degrees C was similar to that of the lymph node lymphocytes (LNL) with maximum after 5 min of incubation and subsequent decrease to 10% of the maximum after 90 min of incubation. Maximal binding for the entire thymocyte population was twice lower than for the cortisone-resistant thymocytes (CRT) or for LNL. Binding of [3H]QNB carried out at 4 degrees C resulted in disappearance of the maximum, but did not alter the difference between CRT and entire thymocyte population. Depletion of CRT detectable [3H]QNB-specific binding to thymocytes until 18th day of gestation but the maximal binding increased up to 20% at the day 19 and reached 90% of adult level on the third day after birth. Moreover, carbamylcholine (a muscarinic agonist) treatment in vivo induced a significant decrease in [3H]QNB binding to the thymocytes. We thus suggest that a subpopulation of thymocytes bearing muscarinic receptors in the periphery acquired these receptors in the thymus as one of the last steps of their maturation. We cannot exclude the possibility that cholinergic stimulation might trigger these lymphocytes to leave the thymus.

Expression of muscarinic cholinergic receptors on lymphocytes during rheumatoid arthritis.

The level of muscarinic receptors on lymphocytes from rheumatoid arthritis (RA) patients in comparison with healthy individuals was determined by the binding studies of a specific muscarinic ligand [3H]-QNB in the presence of the competitive antagonist atropine. The response to phytohaemagglutinin and the influence of acetylcholine on this process in patients' lymphocytes was also studied. We have found direct correlation among PHA stimulation indices, the level of muscarinic receptors, and the influence of acetylcholine on PHA stimulation indices. Differences in these parameters occurring in systemic forms of RA as compared to other RA patients are also described and their possible mechanism discussed. The influence of cholinergic stimulation on the reactivity of lymphocytes after PHA in different forms of RA could depend on the number of muscarinic receptors present on the lymphocyte surface, as well as on the activity of soluble factors, which could modulate the functional state of the receptors.

Muscarinic receptors of rat lymphocytes--differences in young and old animals.

Muscarinic receptors were studied on lymphocytes from young and old Wistar rats. Binding studies were performed by the use of [3H]-QNB, a specific muscarinic antagonist. Some differences between these two groups were observed. Maximal binding of [3H]-QNB and half time of the maximal binding is lower for lymphocytes of old rats [3H]-QNB receptor complexes could not be found in the supernatants derived from lymphocytes of old animals. Higher ability to loose or hide the muscarinic receptors was also observed in this group of rats. All these observations could reflect a more effective degradation, as well as a lower level of muscarinic receptors exposed on lymphocytes from old animals.

Muscarinic acetylcholine receptors of rat lymphocytes.

The muscarinic acetylcholine receptors on rat lymphocytes were determined by [3H]quinuclidinyl benzilate binding studies. Binding of [3H]quinuclidinyl benzilate is rapid (half saturation occurred within 120 s) and highly specific. Muscarinic receptors reveal high lability. The number of receptors on plasma membrane depends on time of incubation as well as on composition of incubation medium. Lymphocytes incubated in nutrient-deficient media lose their surface receptors; enrichment of the medium causes reappearance of the receptors. Appearance of [3H]quinuclidinyl benzilate-binding sites in the incubation medium was under conditions in which binding to lymphocytes was decreased. It is concluded that the number of plasma membrane receptors on rat lymphocytes represents the dynamic steady state in which newly synthesized and degraded receptors are balanced.

Cultured beating myocytes from neonatal rat heart as a model for the study of muscarinic cholinergic receptors.

Neonatal rat heart myocytes were isolated by digestion of the heart tissue with trypsin. After separation the cells were transferred to plastic cluster dishes where they formed a monolayer culture. After 72 hours in culture the myocytes showed a synchronous contraction and renovation of their membrane structures. Studies of muscarinic cholinergic receptors were carried out using (3H)-Quinuclidinyl benzilate directly on cells attached to the bottom of the dish. The presence of a specific muscarinic receptor on cultured beating myocytes was confirmed.

Cholinergic stimulation of RNA synthesis in rat lymphocytes.

In unstimulated lymphocytes acetylcholine caused concentration-dependent increase of RNA synthesis. Atropine and tubocurarine did not inhibit this stimulating effect, but, on the contrary, gave an additional peak of uridine incorporation. In lymphocytes stimulated previously with PHA, acetylcholine produced changes in RNA synthesis which did not depend on the dose of the mitogen. The stimulating effect of acetylcholine was inhibited at all concentrations by tubocurarine, while atropine abolished only the positive effects of higher acetylcholine concentrations. The results suggest occurrence of three different types of acetylcholine action on rat lymphocytes.

Acetylcholine receptors of rat lymphocytes.

The conditions of the binding of acetylcholine have been studied in lymphocytes isolated from rat peripheral lymph nodes. Acetylcholine appeared to penetrate the lymphocyte membrane. We have confirmed the presence of muscarinic receptors, which, however, are not involved in transport of acetylcholine through the membrane. The receptors of the nicotine type on lymphocytes are demonstrated by the decrease of acetylcholine binding in the presence of a specific antagonist, tubocurarine. These nicotinic receptors may be involved in acetylcholine transport into the cells.

Studies on cyclic AMP-cyclic GMP cooperation and on the influence of acetylcholine, adrenaline and thiols in lymphocyte reactivity.

In rat's lymph node lymphocytes, maximal stimulation of the response after suboptimal doses of PHA (1/2 of the optimal dose), measured by the incorporation of 3H-thymidine, after 10(-6) M acetylcholine and after 10 min interval between PHA and acetylcholine application was obtained. In these experiments inhibition of acetylcholine action by its muscarainic antagonists was not observed. It was shown that exogeneous cAMP as well as other factors, which increase cAMP level in lymphocytes had opposite effects to those, which could be caused by increasing of cGMP. Acetylcholine in optimal concentration (10(-6) M) as well as thiols, did not increase the level of endogeneous cAMP in rat lymphocytes.

Precursors of cytoplasmic RNA in avian cell nucleus.

In nucleus of chick embryo four pre-rRNA of molecular weight of 3.43, 2.93, 2.36 and 1.87 X 10(6) were found in addition to the heterogeneous population of "giant" RNA molecules larger than 4.5 X 10(6) daltons, partially polyadenylated, with few methyl groups and numerous double-stranded regions.