Effect of Mycobacterium leprae in peripheral nerve trunk on the evolution of skin lesions.

Comparative histological studies were made of a) 41 peripheral nerve lesions and the skin in the area of supply, and b) 12 peripheral nerve lesions and concurrent but unrelated skin lesions. In the first study, small, relatively early, histologically classifiable skin lesions were found in all cases, even though there were no clinical lesions. In every case the lesion was centered on a dermal nerve. In some cases disruption of the perineurium was associated with emergence of the lesion into the dermis and a small silent local reaction. It was concluded that there was a descending spread of the disease down the neural pathway to the dermis, although it was not necessarily associated with transport of bacilli. Although the first study showed a discrepancy in the classification between skin and nerve lesions in nearly 50% of the cases (as previously reported), the second study showed no discrepancies. It is suggested that discrepancies are relatively uncommon, and that those in the first study are exceptional. The probable explanation is that microreactions in the nerve trunks had caused a shift in classification, which was not yet reflected in the immature skin lesions. In the second study, the mature skin lesions had reached immunological equilibrium. Discrepancies in classification between skin and nerve lesions, as between concurrent skin lesions, are the result of reaction. Attention is drawn to the probable role of subliminal reactions in the evolution of infections.

Positive Mitsuda lepromin reactions in long-term treated lepromatous leprosy.

Twenty-four lepromatous (LL) patients, treated for 22 to 40 years with chemotherapy, including sulphones and with multidrug therapy, were tested with standard Wade-Mitsuda lepromin. Thirteen gave weak positive (3-4 mm) Mitsuda reactions, confirmed histologically in the ten whose reactions were biopsied. Six of the eleven negative reactors were partly accounted for by a history of relapse, and two others had probably taken dapsone irregularly. Eleven control LL patients, treated for less than 20 years, were uniformly lepromin negative. Spontaneous lepromin conversion appears to occur around 24 years after commencing successful chemotherapy. The late Mitsuda conversions are attributed to delayed clearance of the reservoir of bacterial antigen, but a poor correlation between Mitsuda and Fernandez positivity is not explained.

Tuberculoid relapse in lepromatous leprosy.

It is commonly accepted that the attainment of bacteriological negativity fails to restore the immune state of leprosy patients who have downgraded to lepromatous. We report six patients who had been lepromatous (LLs), and who, after many years of chemotherapy and bacteriological negativity, were found upon relapse to have upgraded to borderline-tuberculoid (BT). Five had become Mitsuda lepromin positive. The relapses could be accounted for by proven or suspected dapsone resistance. The upgrading was associated with minimal signs of reaction, which was attributed to the low level of antigen in the almost resolved lesions. The manner of development of the new high immune lesions resembled the onset of a primary infection, clinically and histologically. The development of a positive Mitsuda reaction in longstanding LL leprosy is not necessarily an indication of cure.

Mycobacterium leprae reactive T cell clones from lepromatous leprosy patients after prolonged dapsone chemotherapy.

The proliferative responses of peripheral blood mononuclear cells (PBMC) to Mycobacterium leprae and BCG were studied in two groups of leprosy patients: a group of 8 lepromatous patients who had been on treatment for more than 20 years (TLL) and a group of 8 untreated lepromatous leprosy patients (ULL). The mean response to M. leprae of the TLL group was 6195 cpm with 5 of the 8 patients responding positively. The mean response to M. leprae of the ULL group was 617 cpm, with only 1 patient showing a positive response. The corresponding proliferative responses to BCG were 19,908 cpm in the TLL group and 7908 in the ULL group. Thirteen M. leprae reactive clones were established from 2 TLL patients and 5 M. leprae reactive clones were established from 2 tuberculoid leprosy patients. Seven of these clones, 4 from the TLL patients and 3 from the tuberculoid (TT) patients could be studied further. Three of the TLL clones responded specifically to M. leprae, while one of the clones exhibited a broad cross-reactivity to other mycobacteria. All of these clones were of the CD4+CD8- phenotype. Our findings suggest that responsiveness to M. leprae can be detected in vitro in a proportion of LL patients who have undergone prolonged chemotherapy, and that this response involves M. leprae reactive CD8+CD8- T cells, of which some appear to be specific to M. leprae.

Histological analysis and the pathogenesis of mucocutaneous leishmaniasis.

A series of over 400 well-documented biopsies of mucocutaneous leishmaniasis was evaluated to elucidate the histological processes associated with the elimination of parasites, and their correlation with the course of the disease. Non-specific inflammation was the most frequent and least effective response; its onset might be delayed, and in this event particularly the incidence of metastasis from skin to mucosa was high. Lysis of parasite-laden macrophages appeared to be the basic mechanism of parasite reduction, even when it was not overt. When it was acute the onset was usually rapid, and though it resulted in much tissue destruction the prognosis was generally better and mucosal metastasis rare. Lysis and non-specific inflammation both led to the formation of a post-necrotic type of granuloma, but reversion of the process was almost as common as progression. Ultimately a tuberculoid granuloma evolved and proceeded to resolution. In about 5 per cent of cases, macrophage activation appeared to bring about early resolution; neither reversion nor mucosal metastasis was seen.

Rationale for the histological spectrum of tuberculosis. A basis for classification.

There is need to re-appraise the cellular response to Mycobacterium tuberculosis. Histological analysis of 54 untreated patients with established disease demonstrated a continuous spectrum of tissue responses in which six groups correlated with evidence of resistance to bacterial multiplication. A predominance of cases in the two middle groups (82%) signified an immunological equilibrium in middle grade resistant patients that is absent in related diseases such as leprosy and cutaneous leishmaniasis. The dominant feature was necrosis, which increased progressively across the spectrum. Its form varied from minimal fibrinoid change, through fine eosinophilic necrosis, to basophilic necrosis characterized by neutrophil karyorrhexis, and finally to an almost acellular lesion with many bacilli. Cytological differentiation of the granuloma was of subsidiary significance, mature epithelioid cells being found only in high resistant cases. No correlation was found for the number of lymphocytes. This classification is thought to be an accurate reflection of the immune state in relation to antigenic load. It raises a hitherto unconsidered possibility that "caseation", a loosely applied macroscopic term, may embrace immunologically distinct states. The classification of multiple lesions was consistent. Histology offers a promising basis for further immunopathological investigation.

Events surrounding the recognition of Mycobacterium leprae in nerves.

Histological examination and immunocytochemistry of Schwann cells, macrophages, and mycobacterial antigen were used to study 48 nerves of untreated patients with leprosy. None of the patients was in reaction clinically, but microreactions, involving small clusters of Schwann cells and macrophages in all cases except LL, were marked by progressive degradation of acid-fast bacilli (AFB). This was thought to be the response to the recognition of mycobacterial antigen. In the first phase, the disintegration of one or more Schwann cells caused the release of AFB, accompanied by subacute inflammation. In the second phase, as edema and cellular infiltration subsided, the necrosis of Schwann cells was replaced by granuloma formation, mycobacterial antigen being in a soluble form. Myelinated cells harbored few degraded AFB, and there was evidence that antigen-associated myelin hastened the death of Schwann cells. Only then did antigen become immunologically detectable to induce an inflammatory response whose clearance and resolution was impeded by the restraint on cellular movement due to the structure of neural tissue. These developments were sporadic but continuous. AFB and antigen released by disintegrating Schwann cells were ingested by regenerating Schwann cells and by macrophages, producing a self-perpetuating cycle which might involve either small areas or the greater part of a fascicle, and could conceivably progress to a generalized reaction.

Classification of nerves is modified by the delayed recognition of Mycobacterium leprae.

Biopsies of 42 concurrent nerve and skin lesions across the spectrum of leprosy were classified and compared histologically and bacteriologically. Observations were made as follows: a) The bacterial load was higher in nerve than in skin lesions of the same histological classification, and it was higher in nerve than in concurrent skin lesions irrespective of classification, although not at the lepromatous pole. b) There was some discrepancy between the histological classification of nerve and skin lesions in half the cases. Skin classification appeared to represent the general tissue response and, insofar as discrepancies existed, the skin classification was thought to give the better evaluation. Nerve classification was subject to minor variations of a random nature which were thought to be the outcome of local reactions due to the build up of antigen as a result of delayed recognition in an immunologically protected situation. Upgrading or downgrading ensued locally, depending on the level of antigen at the time of its detection. In such cases, the corresponding skin classification was usually BT, which occupied a critical point in the spectrum. A certain autonomy of the response between lesions of skin and nerve suggests an explanation for downgrading reactions. Although Mycobacterium leprae, alone among mycobacteria, has some sort of affinity for Schwann cells, it is the role of the nerves as protected sites which is fundamental to the course of the disease.

Histochemical demonstration of mycobacterial antigen, specific antibody and complement in the lesions of tuberculosis.

Lesions were studied histochemically for mycobacterial antigen, its specific antibody and complement in 31 patients with recently-diagnosed tuberculosis. The results were related to a histological spectrum that correlated with bacterial load. The form, localization and persistence of antigen were found to be as significant as the amount. In high-resistant cases, the antigen was mainly soluble, a form which was non-toxic when ingested by macrophages but associated with tissue damage when bound to connective tissue. There was no close contact between plasma cells and antigen. However, in cases with moderate resistance, where plasma cells and antigen intermingled freely, necrosis with karyorrhexis and polymorph infiltration was associated with deposition of antigen, antibody and complement at the same sites, indicating the probability of immune complex formation in these lesions. In low-resistant cases, extensive necrosis was attributed partly to high levels of extracellular antigen. The correlation between immunological circumstances and the manifold forms of necrosis validated these forms as the basis for a histological spectrum in tuberculosis.

Monocyte recruitment, antigen degradation and localization in cutaneous leishmaniasis.

The relationship between the destruction of Leishmania, the recruitment of monocytes and macrophage activity in the lesions of cutaneous leishmaniasis (CL) was studied in 53 biopsies representing the phases of evolution of the infection. Lysozyme, amastigotes and their degradation products were located by their specific antibodies. A rising level of monocyte influx was found to correlate with the degradation and solubilization of antigen, a falling level with final clearance. Differences in the results supported the previous concept of macrophage activation and macrophage lysis as alternative mechanisms for the elimination of Leishmania. Macrophage activation appeared to coincide with re-phagocytosis of externalized antigenic products of different type and origin. Macrophage lysis was a fully effective mechanism only when the antigen was contained within a focalized granuloma before mass lysis. Failing this, degradation and clearance of antigen were incomplete, and residues were sequestered on the periphery of the lesion where they bound to collagen and epidermis with consequential tissue damage. Antigen was demonstrated on the surface of lightly parasitized macrophages but not heavily infected ones. Other cells bound antigen without ingesting it, a process which might allow antigen presentation though it would also favour survival of parasites within the cell.

An evaluation of the ELISA for schistosomiasis in a hospital population.

In order to evaluate the ELISA for schistosomiasis under the conditions of clinical practice, 1576 hospital patients were tested using a crude soluble Schistosoma mansoni egg antigen. Test sensitivity in detection of S. mansoni was found to be 96.2% and in S. haematobium 92.3%. The predictive value of positive results was high, reaching 88% at antibody levels three or more times the screening level. The test was considered by clinicians to be valuable for diagnosis and patient management, though it did not distinguish active from recently treated infections. Of 37 apparently false positive schistosome ELISA results only seven could be attributed to other helminth infections. Another nine patients had hepatitis. It is suggested that the antigens and antibodies of the two diseases are mutually cross-reactive, since reports have suggested a high increase of HBsAg patients with schistosomiasis.

Lysozyme as a measure of cellular dynamics in the lesions of leprosy.

The levels and distribution of lysozyme-positive cells and exudate were studied in leprosy lesions through the spectrum, in untreated and treated patients, in relapse and in reactions. Altogether 124 skin biopsies were examined by the immunoperoxidase technique. Monocytes, neutrophil-polymorphs and mast cells were the most conspicuous cells seen. Lysozyme proved to be a useful means of indexing renewal of these cells in the lesions. Peak numbers of monocytes were seen in lesions of active lepromatous leprosy (LL) and of tuberculoid leprosy (TT), at poles of opposite immunological performance. In TT the stimulus for recruitment was delayed hypersensitivity (DH). A decline in DH from TT towards the middle of the spectrum, mid-borderline, was accompanied by a fall in monocyte level. Furthermore, reacting lesions due to enhanced DH also had increased numbers of monocytes. On the other hand reactions associated with immunological deterioration were similar to active lepromatous leprosy (LL) and monocyte influx was raised in response to the stimulus of free multiplication of bacilli in both cases. In TT delayed hypersensitivity acted also to promote the rapid transformation of monocytes to epithelioid and giant cells all of which were strongly positive for lysozyme. This was in contrast to much lower levels in histologically similar macrophage-epithelioid cells of BT granulomas. Lysozyme synthesis was not seen in macrophages after ingestion of M. leprae. Early foamy change was made conspicuous by lysozyme deposited in phagocytic vacuoles, but old foam cells in regressing lepromas were negative. Lysozyme bound to dead extracellular M. leprae but not to viable or intracellular organisms. Dead bacilli or immune complexes appeared to be the stimulus for neutrophil-polymorph recruitment, mainly in reactions.

Exacerbation reactions in hyperactive lepromatous leprosy.

Exacerbation reactions (ER) are acute reactions occurring locally in histoid or other highly active lepromatous lesions with an exceptionally heavy bacterial load. Clinically, they are almost silent although they may cause ulceration and the release of viable bacilli. Histologically, the influx of polymorph neutrophils and coincident macrophage degeneration mimic erythema nodosum leprosum (ENL). Later, the signs of increased permeability or necrosis of small blood vessels and mast cell degranulation are differential features. The predominant immunoglobulin is IgE, and the main complement component is C1q, C3 being mostly undetectable. The reactions appear to be mediated in part by reagins (although eosinophils are not seen). Immune complexes probably form at antigen excess. Of equal importance may be the release from highly activated macrophages and neutrophils of hydrolases and proteases, which are capable of degrading connective tissue and other cell surfaces. This report is based on a histopathological and an immunocytological study of 13 exacerbation reactions in comparison with nonreacting hyperactive lesions and with ENL. The results support the view that the essential feature of histoid lesions is their hyperactivity.

Cutaneous leishmaniasis: immune complex formation and necrosis in the acute phase.

Twenty biopsies of lesions of cutaneous leishmaniasis were classified according to the mechanism of parasite elimination, on the basis of macrophage activation (five cases) or macrophage lysis (15 cases). The immunoperoxidase technique was used to demonstrate free Leishmania antigen, immunoglobulins, complement, lysozyme, C-reactive protein, beta-lipoprotein, alpha 1-antitrypsin, alpha 2-macroglobulin, plasminogen and factor VIII, which were quantitated and comparatively assessed. The fall in the parasite load during the course of the infection was associated with rising levels of IgG, IgM and IgE, and of the complement components of the classical pathway. Macrophage lysis supervened when there was an approximate equivalence of antigen and antibody, and was associated with the deposition of immune complex components. Lysis of the acute focal type (C response) was accompanied by a massive liberation of free Leishmania antigen, followed by a fall indicative of parasite elimination. The lysis of small numbers of macrophages scattered diffusely in the lesion, which was slow to reach completion (B response), was less effective and immunologically closer to the non-lytic (A) response. A terminal fall of the immunological factors other than the globulins, suggestive of resolution, was observed mainly in the C response. Lymphocytes may be important in macrophage activation associated with the macrophage A response and in the later stage of the B and C responses. However immunologically induced host-cell lysis is more important than macrophage activation for the elimination of Leishmania in the acute stage of most skin lesions. It is associated with, and may be caused by, the formation in situ of immune complexes of Leishmania antigen and antibody at an appropriate ratio.

Late-stage cutaneous leishmaniasis: immunopathology of tuberculoid lesions in skin and lymph nodes.

Strong development of tuberculoid features in the late stage of cutaneous leishmaniasis presents immunopathological, diagnostic and sometimes therapeutic problems. Only two such cases were identified out of 30 late-stage cases of the disease. They were delineated on histological and immunocytological grounds, and found to present higher levels of Leishmania antigen and lower levels of IgG than the weakly tuberculoid cases. One case showed histological and skin test evidence of delayed-type hypersensitivity; the other case (clinically of the relapsing or recidiva variety) lacked such evidence. Here the residual antigen was segregated at the periphery of the lesion. In three late-stage cases for which lymph node biopsies were available there was a stronger tuberculoid response in lymph node or lymphatic than in the skin lesion. In two cases there was caseation. Germinal follicles were notably active. It is suggested that in cutaneous leishmaniasis a strong tuberculoid response may be associated not so much with immune complex formation (as in the majority of lesions) but with sequestration of antigen at a peripheral site or in the lymphatic apparatus.

C-reactive protein and apoB containing lipoproteins are associated with Mycobacterium leprae in lesions of human leprosy.

Skin biopsies from patients with leprosy across the spectrum from tuberculoid (TT) to lepromatous (LL), including histoid lepromas and erythema nodosum leprosum (ENL) reactions, were stained immunohistochemically for the presence of C-reactive protein (CRP) and the apolipoprotein, apoB. Mycobacterium leprae bacillary material comprising cell walls, cytoplasmic and soluble components was present with increasing abundance towards the lepromatous end of the spectrum and always stained positively with anti-CRP. M. leprae from armadillos did not stain with anti-human CRP indicating that the staining of M. leprae in human tissues was not due to a cross-reaction between anti-CRP and the organism itself. When CRP was present in large amounts apoB was also demonstrated in the same distribution. CRP was detected on bacilli and their degradation products within the cytoplasm of macrophages even in the absence of a raised serum CRP level in some ENL patients and also in two cases of advanced resolving lepromas. These findings demonstrate remarkable persistence of CRP in association with M. leprae in vivo, and raise intriguing questions about the possible role of CRP in relation to the handling of leprosy bacilli.